Compositions comprising enzyme-cleavable amphetamine prodrugs and inhibitors thereof

ABSTRACT

Pharmaceutical compositions and their methods of use are provided, where the pharmaceutical compositions comprise an amphetamine prodrug that provides enzymatically-controlled release of amphetamine or an amphetamine analog. The composition can further comprise an enzyme inhibitor that interacts with the enzyme(s) that mediates the enzymatically-controlled release of amphetamine or the amphetamine analog from the amphetamine prodrug so as to attenuate enzymatic cleavage of the amphetamine prodrug.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/042,906 filed Feb. 12, 2016, which is a divisional of U.S. patentapplication Ser. No. 13/634,524 filed Nov. 19, 2012, which is a 371 ofPCT Application No. PCT/US2011/031846 filed Apr. 8, 2011, which claimsbenefit to U.S. Provisional Application Ser. No. 61/326,609 filed onApr. 21, 2010, each of which application is incorporated herein byreference in its entirety.

INTRODUCTION

Amphetamines are susceptible to misuse, abuse, or overdose. Use of andaccess to these drugs therefore needs to be controlled. The control ofaccess to these types of drugs is expensive to administer and can resultin denial of treatment for patients suffering from conditions such asAttention Deficit Hyperactivity Disorder (ADHD), Chronic FatigueSyndrome (CFS), brain injury, narcolepsy, or obesity. Furthermore,control of use is often ineffective, leading to substantial morbidityand deleterious social consequences. Current existing amphetamine drugproducts provide no significant barriers to abuse and misuse bypatients.

SUMMARY

The present disclosure provides pharmaceutical compositions, and theirmethods of use, where the pharmaceutical compositions comprise anamphetamine prodrug that provides enzymatically-controlled release ofamphetamine or an amphetamine analog. The composition can furthercomprise an enzyme inhibitor that interacts with the enzyme(s) thatmediates the enzymatically-controlled release of amphetamine or theamphetamine analog from the amphetamine prodrug so as to attenuateenzymatic cleavage of the amphetamine prodrug.

The embodiments include compositions comprising an amphetamine prodrug,wherein the amphetamine prodrug comprises amphetamine or an amphetamineanalog covalently bound to a promoiety comprising a GI enzyme-cleavablemoiety, wherein cleavage of the GI enzyme-cleavable moiety by a GIenzyme mediates release of amphetamine or the amphetamine analog; and,optionally, a GI enzyme inhibitor that interacts with the GI enzyme thatmediates enzymatically-controlled release of amphetamine or theamphetamine analog from the amphetamine prodrug following ingestion ofthe composition. Such cleavage can initiate, contribute to or effectrelease of amphetamine or the amphetamine analog.

The embodiments include dose units comprising compositions comprising anamphetamine prodrug and a GI enzyme inhibitor, where the amphetamineprodrug and GI enzyme inhibitor are present in the dose unit in anamount effective to provide for a pre-selected pharmacokinetic (PK)profile following ingestion. In further embodiments, the pre-selected PKprofile comprises at least one PK parameter value that is less than thePK parameter value of amphetamine released following ingestion of anequivalent dosage of amphetamine prodrug in the absence of inhibitor. Infurther embodiments, the PK parameter value is selected from anamphetamine Cmax value, an amphetamine exposure value, and a(1/amphetamine Tmax) value.

In certain embodiments, the dose unit provides for a pre-selected PKprofile following ingestion of at least two dose units. In relatedembodiments, the pre-selected PK profile of such dose units is modifiedrelative to the PK profile following ingestion of an equivalent dosageof amphetamine prodrug without inhibitor. In related embodiments, such adose unit provides that ingestion of an increasing number of the doseunits provides for a linear PK profile. In related embodiments, such adose unit provides that ingestion of an increasing number of the doseunits provides for a nonlinear PK profile. In related embodiments, thePK parameter value of the PK profile of such a dose units is selectedfrom an amphetamine Cmax value, a (1/amphetamine Tmax) value, and anamphetamine exposure value.

The embodiments include compositions comprising a container suitable forcontaining a composition for administration to a patient; and a doseunit as described herein disposed within the container.

The embodiments include dose units of an amphetamine prodrug and a GIenzyme inhibitor wherein the dose unit has a total weight of from 1microgram to 2 grams. The embodiments include pharmaceuticalcompositions of an amphetamine prodrug and a GI enzyme inhibitor whereinthe combined weight of amphetamine prodrug and GI enzyme inhibitor isfrom 0.1% to 99% per gram of the composition.

The present disclosure provides a compound of formula AM-(I), andcompositions and dose units comprising a compound of formula AM-(I):

wherein

R¹ is selected from hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, and substituted heteroarylalkyl; and

R² is an acyl, substituted acyl, or an N-acyl derivative of a peptide;

or a salt, hydrate or solvate thereof.

The present disclosure provides a compound of formula AM-(II), andcompositions and dose units comprising a compound of formula AM-(II):

wherein

R¹ is selected from hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, and substituted heteroarylalkyl; and

R² is an acyl, substituted acyl, or an N-acyl derivative of a peptide;

or a salt, hydrate or solvate thereof.

The present disclosure provides Compound AM-1, and compositions and doseunits comprising Compound AM-1:

or a salt, hydrate or solvate thereof.

The present disclosure provides Compound AM-2, and compositions and doseunits comprising Compound AM-2:

or a salt, hydrate or solvate thereof.

The present disclosure provides Compound AM-5, and compositions and doseunits comprising Compound AM-5:

or a salt, hydrate or solvate thereof.

The present disclosure provides a compound of formula AM-(III), andcompositions and dose units comprising a compound of formula AM-(III):

wherein

R is a GI enzyme-cleavable moiety.

The present disclosure provides a compound of formula AM-(IV), andcompositions and dose units comprising a compound of formula AM-(IV):

wherein

R is a GI enzyme-cleavable moiety.

The embodiments include methods for treating a patient comprisingadministering any of the compositions or dose units described herein toa patient in need thereof. The embodiments include methods to reduceside effects of a therapy comprising administering any of thecompositions or dose units described herein to a patient in needthereof. The embodiments include methods of improving patient compliancewith a therapy prescribed by a clinician comprising directingadministration of any of the compositions or dose units described hereinto a patient in need thereof. Such embodiments can provide for improvedpatient compliance with a prescribed therapy as compared to patientcompliance with a prescribed therapy using drug and/or using prodrugwithout inhibitor as compared to prodrug with inhibitor.

The embodiments include methods of reducing risk of unintended overdoseof amphetamine comprising directing administration of any of thepharmaceutical compositions or dose units described herein to a patientin need of treatment.

The embodiments include methods of making a dose unit comprisingcombining an amphetamine prodrug and a GI enzyme inhibitor in a doseunit, wherein the amphetamine prodrug and GI enzyme inhibitor arepresent in the dose unit in an amount effective to attenuate release ofamphetamine from the amphetamine prodrug.

The embodiments include methods of deterring misuse or abuse of multipledose units of an amphetamine prodrug comprising combining an amphetamineprodrug and a GI enzyme inhibitor in a dose unit, wherein theamphetamine prodrug and GI enzyme inhibitor are present in the dose unitin an amount effective to attenuate release of amphetamine from theamphetamine prodrug such that ingestion of multiples of dose units by apatient does not provide a proportional release of amphetamine. Infurther embodiments, release of drug is decreased compared to release ofdrug by an equivalent dosage of prodrug in the absence of inhibitor.

One embodiment is a method for identifying a prodrug and a GI enzymeinhibitor suitable for formulation in a dose unit. Such a method can beconducted as, for example, an in vitro assay, an in vivo assay, or an exvivo assay.

The embodiments include methods for identifying an amphetamine prodrugand a GI enzyme inhibitor suitable for formulation in a dose unitcomprising combining an amphetamine prodrug, a GI enzyme inhibitor, anda GI enzyme in a reaction mixture, and detecting amphetamine prodrugconversion, wherein a decrease in amphetamine prodrug conversion in thepresence of the GI enzyme inhibitor as compared to amphetamine prodrugconversion in the absence of the GI enzyme inhibitor indicates theamphetamine prodrug and GI enzyme inhibitor are suitable for formulationin a dose unit.

The embodiments include methods for identifying an amphetamine prodrugand a GI enzyme inhibitor suitable for formulation in a dose unitcomprising administering to an animal an amphetamine prodrug and a GIenzyme inhibitor and detecting amphetamine prodrug conversion, wherein adecrease in amphetamine conversion in the presence of the GI enzymeinhibitor as compared to amphetamine conversion in the absence of the GIenzyme inhibitor indicates the amphetamine prodrug and GI enzymeinhibitor are suitable for formulation in a dose unit. In certainembodiments, administering comprises administering to the animalincreasing doses of inhibitor co-dosed with a selected fixed dose ofamphetamine prodrug. Detecting prodrug conversion can facilitateidentification of a dose of inhibitor and a dose of amphetamine prodrugthat provides for a pre-selected pharmacokinetic (PK) profile. Suchmethods can be conducted as, for example, an in vivo assay or an ex vivoassay.

The embodiments include methods for identifying an amphetamine prodrugand a GI enzyme inhibitor suitable for formulation in a dose unitcomprising administering to an animal tissue an amphetamine prodrug anda GI enzyme inhibitor and detecting amphetamine prodrug conversion,wherein a decrease in amphetamine prodrug conversion in the presence ofthe GI enzyme inhibitor as compared to amphetamine prodrug conversion inthe absence of the GI enzyme inhibitor indicates the amphetamine prodrugand GI enzyme inhibitor are suitable for formulation in a dose unit.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representing the effect of increasing the level ofa GI enzyme inhibitor (“inhibitor”, X-axis) on a PK parameter (e.g.,drug Cmax) (Y-axis) for a fixed dose of prodrug. The effect of inhibitorupon a prodrug PK parameter can range from undetectable, to moderate, tocomplete inhibition (i.e., no detectable drug release).

FIG. 2 provides schematics of drug concentration in plasma (Y-axis) overtime (X-axis). Panel A is a schematic of a pharmacokinetic (PK) profilefollowing ingestion of prodrug with a GI enzyme inhibitor (dashed line)where the drug Cmax is modified relative to that of prodrug withoutinhibitor (solid line). Panel B is a schematic of a PK profile followingingestion of prodrug with inhibitor (dashed line) where drug Cmax anddrug Tmax are modified relative to that of prodrug without inhibitor(solid line). Panel C is a schematic of a PK profile following ingestionof prodrug with inhibitor (dashed line) where drug Tmax is modifiedrelative to that of prodrug without inhibitor (solid line).

FIG. 3 provides schematics representing differential concentration-dosePK profiles that can result from the dosing of multiples of a dose unit(X-axis) of the present disclosure. Different PK profiles (asexemplified herein for a representative PK parameter, drug Cmax(Y-axis)) can be provided by adjusting the relative amount of prodrugand GI enzyme inhibitor contained in a single dose unit or by using adifferent prodrug or inhibitor in the dose unit.

FIGS. 4(a) and 4(b) provide graphs of amphetamine exposure results forrats administered with different doses of Compound AM-1 according toembodiments of the present disclosure. FIG. 4(a) shows a graph of meanplasma concentrations over time of amphetamine release following POadministration of increasing doses of Compound AM-1. FIG. 4(b) shows agraph of mean plasma concentrations over time of prodrug disappearanceand of amphetamine release following PO administration of Compound AM-1.

FIG. 5 provides a graph of mean plasma concentrations over time ofamphetamine release following PO administration of Compound AM-1 with orwithout a co-dose of trypsin inhibitor according to embodiments of thepresent disclosure.

FIG. 6 provides a graph of mean plasma concentrations over time ofCompound AM-1 and amphetamine following IV administration of CompoundAM-1 to rats according to embodiments of the present disclosure.

FIGS. 7(a) and 7(b) provide graphs of amphetamine exposure results forrats administered with different doses of Compound AM-2 according toembodiments of the present disclosure. FIG. 7(a) shows a graph of meanplasma concentrations over time of amphetamine release following POadministration of increasing doses of Compound AM-2. FIG. 7(b) shows agraph of mean plasma concentrations over time of prodrug disappearanceand of amphetamine release following PO administration of Compound AM-2.

FIG. 8 shows a graph of mean plasma concentrations over time ofamphetamine release following PO administration of Compound AM-2 with orwithout a co-dose of trypsin inhibitor according to embodiments of thepresent disclosure.

FIG. 9 shows a graph of mean plasma concentrations over time of CompoundAM-2 and amphetamine following IV administration of Compound AM-2 torats according to embodiments of the present disclosure.

FIG. 10 shows a graph of mean plasma concentrations over time of prodrugdisappearance and of amphetamine release following PO administration ofCompound AM-3 according to embodiments of the present disclosure.

FIG. 11 shows a graph of mean plasma concentrations over time ofCompound AM-3 and amphetamine following IV administration of CompoundAM-3 to rats according to embodiments of the present disclosure.

FIG. 12 provides a graph that compares mean plasma concentrations overtime of amphetamine release following PO administration to rats ofprodrug Compound AM-1 co-dosed with increasing amounts of trypsininhibitor Compound 109.

FIG. 13A provides a graph that compares mean plasma concentrations overtime of amphetamine release following PO administration to rats ofsingle and multiple doses of prodrug Compound AM-1 in the absence oftrypsin inhibitor. FIG. 13B provides a graph that compares mean plasmaconcentrations over time of amphetamine release following POadministration to rats of single and multiple dose units comprisingprodrug Compound AM-1 and trypsin inhibitor Compound 109.

FIG. 14 provides a graph of mean plasma concentrations over time ofamphetamine release following PO administration to rats of prodrugCompound AM-5.

FIG. 15 provides a graph of mean plasma concentrations over time ofamphetamine release following PO administration to rats of CompoundAM-4.

FIG. 16 provides a graph of mean plasma concentrations over time ofprodrug Compound AM-5 and amphetamine following IV administration torats of prodrug Compound AM-5.

DEFINITIONS

The following terms have the following meaning unless otherwiseindicated. Any undefined terms have their art recognized meanings.

As used herein, the term “alkyl” by itself or as part of anothersubstituent refers to a saturated branched or straight-chain monovalenthydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane. Typical alkyl groups include, butare not limited to, methyl; ethyl, propyls such as propan-1-yl orpropan-2-yl; and butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl or 2-methyl-propan-2-yl. In some embodiments, analkyl group comprises from 1 to 20 carbon atoms. In other embodiments,an alkyl group comprises from 1 to 10 carbon atoms. In still otherembodiments, an alkyl group comprises from 1 to 6 carbon atoms, such asfrom 1 to 4 carbon atoms.

“Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl radical derived bythe removal of one hydrogen atom from a single carbon atom of an alkane.Typical alkanyl groups include, but are not limited to, methanyl;ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl),cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl(sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl(t-butyl), cyclobutan-1-yl, etc.; and the like.

“Alkylene” refers to a branched or unbranched saturated hydrocarbonchain, usually having from 1 to 40 carbon atoms, more usually 1 to 10carbon atoms and even more usually 1 to 6 carbon atoms. This term isexemplified by groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—),the propylene isomers (e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl radical having atleast one carbon-carbon double bond derived by the removal of onehydrogen atom from a single carbon atom of an alkene. The group may bein either the cis or trans conformation about the double bond(s).Typical alkenyl groups include, but are not limited to, ethenyl;propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl(allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl;butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl,cyclobuta-1,3-dien-1-yl, etc.; and the like.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl radical having atleast one carbon-carbon triple bond derived by the removal of onehydrogen atom from a single carbon atom of an alkyne. Typical alkynylgroups include, but are not limited to, ethynyl; propynyls such asprop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

“Acyl” by itself or as part of another substituent refers to a radical—C(O)R³⁰, where R³⁰ is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl,aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as definedherein and substituted versions thereof. Representative examplesinclude, but are not limited to formyl, acetyl, cyclohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, piperonyl, succinyl,and malonyl, and the like.

The term “aminoacyl” refers to the group —C(O)NR²¹R²², wherein R²¹ andR²² independently are selected from the group consisting of hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic andwhere R²¹ and R²² are optionally joined together with the nitrogen boundthereto to form a heterocyclic or substituted heterocyclic group, andwherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Alkoxy” by itself or as part of another substituent refers to a radical—OR³¹ where R³¹ represents an alkyl or cycloalkyl group as definedherein. Representative examples include, but are not limited to,methoxy, ethoxy, propoxy, butoxy, cyclohexyloxy and the like.

“Alkoxycarbonyl” by itself or as part of another substituent refers to aradical —C(O)OR³¹ where R³¹ represents an alkyl or cycloalkyl group asdefined herein. Representative examples include, but are not limited to,methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,cyclohexyloxycarbonyl and the like.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of an aromatic ring system.Typical aryl groups include, but are not limited to, groups derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene and the like. In certain embodiments, an aryl groupcomprises from 6 to 20 carbon atoms. In certain embodiments, an arylgroup comprises from 6 to 12 carbon atoms. Examples of an aryl group arephenyl and naphthyl.

“Arylalkyl” by itself or as part of another substituent refers to anacyclic alkyl radical in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylalkenyl and/orarylalkynyl is used. In certain embodiments, an arylalkyl group is(C₇-C₃₀) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C₁-C₁₀) and the aryl moiety is (C₆-C₂₀). In certainembodiments, an arylalkyl group is (C₇-C₂₀) arylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C₁-C₈) andthe aryl moiety is (C₆-C₁₂).

“Arylaryl” by itself or as part of another substituent, refers to amonovalent hydrocarbon group derived by the removal of one hydrogen atomfrom a single carbon atom of a ring system in which two or moreidentical or non-identical aromatic ring systems are joined directlytogether by a single bond, where the number of such direct ringjunctions is one less than the number of aromatic ring systems involved.Typical arylaryl groups include, but are not limited to, biphenyl,triphenyl, phenyl-napthyl, binaphthyl, biphenyl-napthyl, and the like.When the number of carbon atoms in an arylaryl group is specified, thenumbers refer to the carbon atoms comprising each aromatic ring. Forexample, (C₅-C₁₄) arylaryl is an arylaryl group in which each aromaticring comprises from 5 to 14 carbons, e.g., biphenyl, triphenyl,binaphthyl, phenylnapthyl, etc. In certain embodiments, each aromaticring system of an arylaryl group is independently a (C₅-C₁₄) aromatic.In certain embodiments, each aromatic ring system of an arylaryl groupis independently a (C₅-C₁₀) aromatic. In certain embodiments, eacharomatic ring system is identical, e.g., biphenyl, triphenyl,binaphthyl, trinaphthyl, etc.

“Cycloalkyl” by itself or as part of another substituent refers to asaturated or unsaturated cyclic alkyl radical. Where a specific level ofsaturation is intended, the nomenclature “cycloalkanyl” or“cycloalkenyl” is used. Typical cycloalkyl groups include, but are notlimited to, groups derived from cyclopropane, cyclobutane, cyclopentane,cyclohexane and the like. In certain embodiments, the cycloalkyl groupis (C₃-C₁₀) cycloalkyl. In certain embodiments, the cycloalkyl group is(C₃-C₇) cycloalkyl.

“Cycloheteroalkyl” or “heterocyclyl” by itself or as part of anothersubstituent, refers to a saturated or unsaturated cyclic alkyl radicalin which one or more carbon atoms (and any associated hydrogen atoms)are independently replaced with the same or different heteroatom.Typical heteroatoms to replace the carbon atom(s) include, but are notlimited to, N, P, O, S, Si, etc. Where a specific level of saturation isintended, the nomenclature “cycloheteroalkanyl” or “cycloheteroalkenyl”is used. Typical cycloheteroalkyl groups include, but are not limitedto, groups derived from epoxides, azirines, thiiranes, imidazolidine,morpholine, piperazine, piperidine, pyrazolidine, pyrrolidine,quinuclidine and the like.

“Heteroalkyl, Heteroalkanyl, Heteroalkenyl and Heteroalkynyl” bythemselves or as part of another substituent refer to alkyl, alkanyl,alkenyl and alkynyl groups, respectively, in which one or more of thecarbon atoms (and any associated hydrogen atoms) are independentlyreplaced with the same or different heteroatomic groups. Typicalheteroatomic groups which can be included in these groups include, butare not limited to, —O—, —S—, —S—S—, —O—S—, —NR³⁷R³⁸—, .═N—N═, —N═N—,—N═N—NR³⁹R⁴⁰, —PR⁴¹—, —P(O)₂—, —POR⁴²—, —O—P(O)₂—, —S—O—, —S—(O)—,—SO₂—, —SnR⁴³R⁴⁴— and the like, where R³⁷, R³⁸, R³⁹, R⁴⁰, R⁴¹, R⁴², R⁴³and R⁴⁴ are independently hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl,substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl or substituted heteroarylalkyl.

“Heteroaryl” by itself or as part of another substituent, refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a heteroaromatic ring system. Typicalheteroaryl groups include, but are not limited to, groups derived fromacridine, arsindole, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene,benzodioxole and the like. In certain embodiments, the heteroaryl groupis from 5-20 membered heteroaryl. In certain embodiments, the heteroarylgroup is from 5-10 membered heteroaryl. In certain embodiments,heteroaryl groups are those derived from thiophene, pyrrole,benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole,oxazole and pyrazine.

“Heteroarylalkyl” by itself or as part of another substituent, refers toan acyclic alkyl radical in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced with aheteroaryl group. Where specific alkyl moieties are intended, thenomenclature heteroarylalkanyl, heteroarylalkenyl and/orheterorylalkynyl is used. In certain embodiments, the heteroarylalkylgroup is a 6-30 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is 1-10 membered and theheteroaryl moiety is a 5-20-membered heteroaryl. In certain embodiments,the heteroarylalkyl group is 6-20 membered heteroarylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-8membered and the heteroaryl moiety is a 5-12-membered heteroaryl.

“Aromatic Ring System” by itself or as part of another substituent,refers to an unsaturated cyclic or polycyclic ring system having aconjugated π electron system. Specifically included within thedefinition of “aromatic ring system” are fused ring systems in which oneor more of the rings are aromatic and one or more of the rings aresaturated or unsaturated, such as, for example, fluorene, indane,indene, phenalene, etc. Typical aromatic ring systems include, but arenot limited to, aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, coronene, fluoranthene,fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene,indane, indene, naphthalene, octacene, octaphene, octalene, ovalene,penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene,phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene,triphenylene, trinaphthalene and the like.

“Heteroaromatic Ring System” by itself or as part of anothersubstituent, refers to an aromatic ring system in which one or morecarbon atoms (and any associated hydrogen atoms) are independentlyreplaced with the same or different heteroatom. Typical heteroatoms toreplace the carbon atoms include, but are not limited to, N, P, O, S,Si, etc. Specifically included within the definition of “heteroaromaticring systems” are fused ring systems in which one or more of the ringsare aromatic and one or more of the rings are saturated or unsaturated,such as, for example, arsindole, benzodioxan, benzofuran, chromane,chromene, indole, indoline, xanthene, etc. Typical heteroaromatic ringsystems include, but are not limited to, arsindole, carbazole,β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene and the like.

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or different substituent(s).Typical substituents include, but are not limited to, alkylenedioxy(such as methylenedioxy), -M, —R⁶⁰, —O—, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R⁶⁰, —OS(O)₂O⁻, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O⁻),—OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹,—C(O)O—, —C(S)OR⁶⁰, —NR⁶²C(O)NR⁶⁰R⁶¹, —NR⁶²C(S)NR⁶⁰R⁶¹,—NR⁶²C(NR⁶³)NR⁶⁰R⁶¹ and —C(NR⁶²)NR⁶⁰R⁶¹ where M is halogen; R⁶⁰, R⁶¹,R⁶² and R⁶³ are independently hydrogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl,heteroaryl or substituted heteroaryl, or optionally R⁶⁰ and R⁶¹ togetherwith the nitrogen atom to which they are bonded form a cycloheteroalkylor substituted cycloheteroalkyl ring; and R⁶⁴ and R⁶⁵ are independentlyhydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl,substituted aryl, heteroaryl or substituted heteroaryl, or optionallyR⁶⁴ and R⁶⁵ together with the nitrogen atom to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certainembodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰, —S—, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂R⁶⁰,—OS(O)₂O⁻, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹),—C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻, —NR⁶²C(O)NR⁶⁰R⁶¹.In certain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —P(O)(OR⁶⁰)(O⁻),—OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻. Incertain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰,—C(O)OR⁶⁰, —C(O)O⁻, where R⁶⁰, R⁶¹ and R⁶² are as defined above. Forexample, a substituted group may bear a methylenedioxy substituent orone, two, or three substituents selected from a halogen atom, a(1-4C)alkyl group and a (1-4C)alkoxy group.

“Dose unit” as used herein refers to a combination of a GIenzyme-cleavable prodrug (e.g., trypsin-cleavable prodrug) and a GIenzyme inhibitor (e.g., a trypsin inhibitor). A “single dose unit” is asingle unit of a combination of a GI enzyme-cleavable prodrug (e.g.,trypsin-cleavable prodrug) and a GI enzyme inhibitor (e.g., trypsininhibitor), where the single dose unit provide a therapeuticallyeffective amount of drug (i.e., a sufficient amount of drug to effect atherapeutic effect, e.g., a dose within the respective drug'stherapeutic window, or therapeutic range). “Multiple dose units” or“multiples of a dose unit” or a “multiple of a dose unit” refers to atleast two single dose units.

“PK profile” refers to a profile of drug concentration in blood orplasma. Such a profile can be a relationship of drug concentration overtime (i.e., a “concentration-time PK profile”) or a relationship of drugconcentration versus number of doses ingested (i.e., a“concentration-dose PK profile”). A PK profile is characterized by PKparameters.

“PK parameter” refers to a measure of drug concentration in blood orplasma, such as: 1) “drug Cmax”, the maximum concentration of drugachieved in blood or plasma; 2) “drug Tmax”, the time elapsed followingingestion to achieve Cmax; and 3) “drug exposure”, the totalconcentration of drug present in blood or plasma over a selected periodof time, which can be measured using the area under the curve (AUC) of atime course of drug release over a selected period of time (t).Modification of one or more PK parameters provides for a modified PKprofile.

“Pharmacodynamic (PD) profile” refers to a profile of the efficacy of adrug in a patient (or subject or user), which is characterized by PDparameters. “PD parameters” include “drug Emax” (the maximum drugefficacy), “drug EC50” (the concentration of drug at 50% of the Emax)and side effects.

“Gastrointestinal enzyme” or “GI enzyme” refers to an enzyme located inthe gastrointestinal (GI) tract, which encompasses the anatomical sitesfrom mouth to anus. Trypsin is an example of a GI enzyme.

“Gastrointestinal enzyme-cleavable moiety” or “GI enzyme-cleavablemoiety” refers to a group comprising a site susceptible to cleavage by aGI enzyme. For example, a “trypsin-cleavable moiety” refers to a groupcomprising a site susceptible to cleavage by trypsin.

“Gastrointestinal enzyme inhibitor” or “GI enzyme inhibitor” refers toany agent capable of inhibiting the action of a gastrointestinal enzymeon a substrate. The term also encompasses salts of gastrointestinalenzyme inhibitors. For example, a “trypsin inhibitor” refers to anyagent capable of inhibiting the action of trypsin on a substrate.

“Pharmaceutical composition” refers to at least one compound and canfurther comprise a pharmaceutically acceptable carrier, with which thecompound is administered to a patient.

“Pharmaceutically acceptable salt” refers to a salt of a compound, whichpossesses the desired pharmacological activity of the compound. Suchsalts include: (1) acid addition salts, formed with inorganic acids suchas hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the compound is replacedby a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, N-methylglucamine and the like.

The term “solvate” as used herein refers to a complex or aggregateformed by one or more molecules of a solute, e.g. a prodrug or apharmaceutically-acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include by way of example, water, methanol, ethanol,isopropanol, acetic acid, and the like. When the solvent is water, thesolvate formed is a hydrate.

“Pharmaceutically acceptable carrier” refers to a diluent, adjuvant,excipient or vehicle with, or in which a compound is administered.

“Preventing” or “prevention” or “prophylaxis” refers to a reduction inrisk of occurrence of a condition, such as ADHD, CFS, brain injury,narcolepsy, or obesity.

“Prodrug” refers to a derivative of an active agent that requires atransformation within the body to release the active agent. In certainembodiments, the transformation is an enzymatic transformation. Prodrugsare frequently, although not necessarily, pharmacologically inactiveuntil converted to the active agent.

“Promoiety” refers to a form of protecting group that when used to maska functional group within an active agent converts the active agent intoa prodrug. Typically, the promoiety will be attached to the drug viabond(s) that are cleaved by enzymatic or non-enzymatic means in vivo.

“Treating” or “treatment” of any condition, such as ADHD, CFS, braininjury, narcolepsy, or obesity, refers, in certain embodiments, toameliorating the condition (i.e., arresting or reducing the developmentof the condition). In certain embodiments “treating” or “treatment”refers to ameliorating at least one physical parameter, which may not bediscernible by the patient. In certain embodiments, “treating” or“treatment” refers to inhibiting the condition, either physically,(e.g., stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both. In certain embodiments,“treating” or “treatment” refers to delaying the onset of the condition.

“Therapeutically effective amount” means the amount of a compound (e.g.prodrug) that, when administered to a patient for preventing or treatinga condition such as ADHD, CFS, brain injury, narcolepsy, or obesity, issufficient to effect such treatment. The “therapeutically effectiveamount” will vary depending on the compound, the condition and itsseverity and the age, weight, etc., of the patient.

DETAILED DESCRIPTION

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It should be understood that as used herein, the term “a” entity or “an”entity refers to one or more of that entity. For example, a compoundrefers to one or more compounds. As such, the terms “a”, “an”, “one ormore” and “at least one” can be used interchangeably. Similarly theterms “comprising”, “including” and “having” can be usedinterchangeably.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

Except as otherwise noted, the methods and techniques of the presentembodiments are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, NewYork: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith andMarch, March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Fifth Edition, Wiley-Interscience, 2001.

The nomenclature used herein to name the subject compounds isillustrated in the Examples herein. In certain instances, thisnomenclature has been derived using the commercially-available AutoNomsoftware (MDL, San Leandro, Calif.).

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the chemical groups represented by the variables arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace compounds thatare stable compounds (i.e., compounds that can be isolated,characterised, and tested for biological activity). In addition, allsub-combinations of the chemical groups listed in the embodimentsdescribing such variables are also specifically embraced by the presentinvention and are disclosed herein just as if each and every suchsub-combination of chemical groups was individually and explicitlydisclosed herein.

General Synthetic Procedures

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any of the means knownin the art, including chromatographic means, such as high performanceliquid chromatography (HPLC), preparative thin layer chromatography,flash column chromatography and ion exchange chromatography. Anysuitable stationary phase can be used, including normal and reversedphases as well as ionic resins. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl,Springer-Verlag, New York, 1969.

During any of the processes for preparation of the compounds of thepresent disclosure, it may be necessary and/or desirable to protectsensitive or reactive groups on any of the molecules concerned. This canbe achieved by means of conventional protecting groups as described instandard works, such as T. W. Greene and P. G. M. Wuts, “ProtectiveGroups in Organic Synthesis”, Fourth edition, Wiley, New York 2006. Theprotecting groups can be removed at a convenient subsequent stage usingmethods known from the art.

The compounds described herein can contain one or more chiral centersand/or double bonds and therefore, can exist as stereoisomers, such asdouble-bond isomers (i.e., geometric isomers), enantiomers ordiastereomers. Accordingly, all possible enantiomers and stereoisomersof the compounds including the stereoisomerically pure form (e.g.,geometrically pure, enantiomerically pure or diastereomerically pure)and enantiomeric and stereoisomeric mixtures are included in thedescription of the compounds herein. Enantiomeric and stereoisomericmixtures can be resolved into their component enantiomers orstereoisomers using separation techniques or chiral synthesis techniqueswell known to the skilled artisan. The compounds can also exist inseveral tautomeric forms including the enol form, the keto form andmixtures thereof. Accordingly, the chemical structures depicted hereinencompass all possible tautomeric forms of the illustrated compounds.The compounds described also include isotopically labeled compoundswhere one or more atoms have an atomic mass different from the atomicmass conventionally found in nature. Examples of isotopes that can beincorporated into the compounds disclosed herein include, but are notlimited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, etc. Compounds canexist in unsolvated forms as well as solvated forms, including hydratedforms. In general, compounds can be hydrated or solvated. Certaincompounds can exist in multiple crystalline or amorphous forms. Ingeneral, all physical forms are equivalent for the uses contemplatedherein and are intended to be within the scope of the presentdisclosure.

Representative Embodiments

Reference will now be made in detail to various embodiments. It will beunderstood that the invention is not limited to these embodiments. Tothe contrary, it is intended to cover alternatives, modifications, andequivalents as may be included within the spirit and scope of theallowed claims.

The present disclosure provides pharmaceutical compositions, and theirmethods of use, where the pharmaceutical compositions comprise anamphetamine prodrug that provides enzymatically-controlled release ofamphetamine or an amphetamine analog. The composition can furthercomprise an enzyme inhibitor that interacts with the enzyme(s) thatmediates the enzymatically-controlled release of amphetamine or theamphetamine analog from the prodrug so as to attenuate enzymaticcleavage of the prodrug. In certain embodiments, the disclosure providespharmaceutical compositions which comprise an enzyme inhibitor and anamphetamine prodrug that contains an enzyme-cleavable moiety that, whencleaved, releases amphetamine or the amphetamine analog.

According to one aspect, the embodiments include pharmaceuticalcompositions, which comprise a gastrointestinal GI enzyme-cleavableamphetamine prodrug and, optionally, a GI enzyme inhibitor. A “GIenzyme-cleavable amphetamine prodrug” is an amphetamine prodrug thatcomprises a promoiety comprising a GI enzyme-cleavable moiety. A GIenzyme-cleavable moiety has a site that is susceptible to cleavage by aGI enzyme. Examples of amphetamine prodrugs and enzyme inhibitors aredescribed below.

Amphetamine Prodrugs

Amphetamine refers to a chemical substance that exerts itspharmacological action by modulating neurotransmitters, such asdopamine, serotonin and norepinephrine. In certain embodiments,amphetamine is a compound with a pharmacophore that crosses theblood-brain barrier and has CNS stimulation and central appetitesuppressant effects. See, for example, Foye's Principles of MedicinalChemistry, Sixth Edition, ed. T. L. Lemke and D. A. Williams, LippincottWilliams & Wilkins, 2008, particularly Chapter 13, pages 392-416.

The present disclosure provides an amphetamine prodrug which providesenzymatically-controlled release of amphetamine. The disclosure providesa promoiety that is attached to amphetamine through the amphetamineamino group.

“Amino-containing amphetamine analogs” or amphetamine analogs” refer toanalogs or derivatives of amphetamine that contain an amino group. Forinstance, the following amphetamine analogs contain an amino group thatcan be a point of attachment to a promoiety through the amino group:Benzedrine (i.e., dl-amphetamine), dextroamphetamine (i.e.,d-amphetamine), levoamphetamine (i.e., 1-amphetamine),4-fluoroamphetamine (4-FA), 3-fluoroamphetamine (3-FA),2-fluoroamphetamine (2-FA), 4-methylthioamphetamine (4-MTA),3,4-methylenedioxyamphetamine (MDA), para-methoxyamphetamine (PMA),3-methoxyamphetamine (3-MeOA), 4-ethoxyamphetamine (4-ETA),2,5-dimethoxy-4-ethoxyamphetamine (MEM),2,5-dimethoxy-4-propoxyamphetamine (MPM), 4-methylamphetamine (4-MA),2-methylamphetamine (2-MA), 3-methylamphetamine (3-MA),3,4-dimethylamphetamine, 3-methoxy-4-methylamphetamine (MMA),3-trifluoromethylamphetamine, 3-hydroxyamphetamine,4-hydroxyamphetamine, (1R,2S)-3-[-2-amino-1-hydroxy-propyl]phenol,2,5-dimethoxy-4-methylamphetamine (DOM),2,6-dimethoxy-4-methylamphetamine (Ψ-DOM), indanylamphetamine,5-(2-aminopropyl)-2,3-dihydrobenzofuran (5-APDB),6-(2-aminopropyl)-2,3-dihydrobenzofuran (6-APDB),5-(2-aminopropyl)indole (5-IT), naphthylaminopropane (NAP),phenylpropanolamine (PPA), d-norpseudoephedrine, benzoylethanamine,para-bromoamphetamine (PBA), para-chloroamphetamine (PCA),para-iodoamphetamine (PIA), α,β-dimethylamphetamine,o-chloro-α,α-dimethylphenethylamine, 3,4-dihydroxyamphetamine (3,4-DHA),2,4-dimethoxyamphetamine (2,4-DMA), 2,5-dimethoxyamphetamine (2,5-DMA),3,4-dimethoxyamphetamine (3,4-DMA), α-methylnorepinephrine (α-Me-NE),2,5-dimethoxy-4-methylthioamphetamine (Aleph),2,5-dimethoxy-4-ethylthioamphetamine (Aleph-2),2,5-dimethoxy-4-isopropylthioamphetamine (Aleph-4),2,5-dimethoxy-4-phenylthioamphetamine (Aleph-6),2,5-dimethoxy-4-propylthioamphetamine (Aleph-7),2,5-dimethoxybromoamphetamine (DOB), 2,5-dimethoxychloroamphetamine(DOC), 2,5-dimethoxyfluoroethylamphetamine (DOEF)2,5-dimethoxyethylamphetamine (DOET), 2,5-dimethoxyfluoroamphetamine(DOF), 2,5-dimethoxyiodoamphetamine (DOI), 2,5-dimethoxynitroamphetamine(DON), 2,5-dimethoxypropylamphetamine (DOPR),2,5-dimethoxytrifluoromethylamphetamine (DOTFM),2-methyl-3,4-methylenedioxyamphetamine (2-methyl-MDA),3-methyl-4,5-methylenedioxyamphetamine (5-methyl-MDA),3-methoxy-4,5-methylenedioxyamphetamine (MMDA),2-methoxy-4,5-methylenedioxyamphetamine (MMDA-2),2-methoxy-3,4-methylenedioxyamphetamine (MMDA-3a),4-methoxy-2,3-methylenedioxyamphetamine (MMDA-3b),2-methylthio-3,4-methylenethioxyamphetamine (2T-MMDA-3a),2-methoxy-4,5-methylenethioxyamphetamine (4T-MMDA-2),3,4,5-trimethoxyamphetamine (TMA), 2,4,5-trimethoxyamphetamine (TMA-2),2,3,4-trimethoxyamphetamine (TMA-3), 2,3,5-trimethoxyamphetamine(TMA-4), 2,3,6-trimethoxyamphetamine (TMA-5),2,4,6-trimethoxyamphetamine (TMA-6),2,5-dimethoxy-3,4-dimethylamphetamine,2,5-dimethoxy-3,4-methylenedioxyamphetamine (DMMDA), tyramine,phentermine, alpha-allyl-phenethylamine, (1-(8-bromobenzo[1,2-b;4,5-b]difuran-4-yl)-2-aminopropane (bromo-DragonFLY),3,4,5-trimethoxyphenethylamine (mescaline),2,5-dimethoxy-4-bromophenethylamine (2C-B),2,5-dimethoxy-4-chlorophenethylamine (2C-C),2,5-dimethoxy-4-iodophenethylamine (2C-I),2,5-dimethoxy-4-methyl-phenethylamine (2C-D),2,5-dimethoxy-4-ethylphenethylamine (2C-E),2,5-dimethoxy-4-n-propylphenethylamine (2C-P),2,5-dimethoxy-4-fluorophenethylamine (2C-F),2,5-dimethoxy-4-nitrophenethylamine (2C-N),2,5-dimethoxy-4-ethylthio-phenethylamine (2C-T-2),2,5-dimethoxy-4-isopropylthio-phenethylamine (2C-T-4),2,5-dimethoxy-4-propylthio-phenethylamine (2C-T-7),2,5-dimethoxy-4-cyclopropylmethylthio-phenethylamine (2C-T-8),2,5-dimethoxy-4-tert-butylthio-phenethylamine (2C-T-9),2,5-dimethoxy-4-(2-fluoroethylthio)-phenethylamine (2C-T-21), ephedrine,pseudoephedrine, and the like.

Any type of reactive group on an amphetamine analog can provide a handlefor a point of attachment to a promoiety. Examples of reactive groups onan amphetamine analog include, but are not limited to, amino, amide,alcohol (including phenol), and ketone. In certain embodiments, an aminogroup on an amphetamine analog provides a point of attachment to apromoiety by reaction to form an amino linkage or an amide. For example,the amino group of the amphetamine analog can provide a point ofattachment to a promoiety by reaction to form an amino linkage or anamide. An amide on an amphetamine analog can provide a point ofattachment to a promoiety by reaction to form a linkage, such as anamide enol or an N-acylated amide. An alcohol (e.g., phenol) on anamphetamine analog can provide a point of attachment to a promoiety byreaction to form a linkage, such as a carbamate, a carbonate, an ether,or an ester. A ketone on an amphetamine analog can provide a point ofattachment to a promoiety by reaction to form a linkage, such as an enolcarbamate.

It is contemplated that amphetamine analogs bearing at least some of thefunctionalities described herein will be developed; such amphetaminesare included as part of the scope of this disclosure.

The disclosure provides for an amphetamine prodrug, wherein amphetamineor the amphetamine analog has an optionally substituted amphetamineresidue of the following general structure:

In certain embodiments, a promoiety can be attached to amphetamine orthe amphetamine analog via modification of the amino moiety of theamphetamine residue. Release of amphetamine or the amphetamine analog ismediated by enzymatic cleavage of the promoiety from amphetamine or theamphetamine analog. In certain embodiments, a promoiety can be attachedto amphetamine or the amphetamine analog through the amino moiety of theamphetamine residue, such as via a covalent bond. Release of amphetamineor the amphetamine analog is mediated by enzymatic cleavage of thepromoiety from amphetamine or the amphetamine analog. In some cases, thepromoiety comprises an enzyme-cleavable moiety that is susceptible tocleavage by a GI enzyme. Such cleavage can initiate, contribute to oreffect drug release.

The disclosure provides an amphetamine prodrug which providesenzymatically-controlled release of amphetamine or an amphetamineanalog. In an amphetamine prodrug, a promoiety is attached viamodification of the amino moiety of the amphetamine residue, such asthrough an amino linkage or as an amide. Release of amphetamine or theamphetamine analog is mediated by enzymatic cleavage of the promoietyfrom amphetamine or the amphetamine analog. The disclosure provides forrelease of amphetamine or the amphetamine analog through enzyme cleavageof the promoiety from amphetamine or the amphetamine analog.

Formula AM-(I)

The present disclosure provides amphetamine prodrugs in which thepromoiety is attached through the amino group of amphetamine. Thedisclosure provides compounds of the general formula AM-(I):

wherein

R¹ is selected from hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, and substituted heteroarylalkyl; and

R² is an acyl, substituted acyl, or an N-acyl derivative of a peptide;

or a salt, hydrate or solvate thereof.

Formula AM-(II)

The embodiments include a compound of formula AM-(II):

wherein

R¹ is selected from hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, and substituted heteroarylalkyl; and

R² is an acyl, substituted acyl, or an N-acyl derivative of a peptide;

or a salt, hydrate or solvate thereof.

In formulae AM-(I) and AM-(II), R¹ is selected from hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl.

In certain instances, in formulae AM-(I) and AM-(II), R¹ is a side chainof an amino acid, such as alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine or valine. In certain instances, R¹ is a side chainof an L-amino acid, such as L-alanine, L-arginine, L-asparagine,L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine,L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine or L-valine.

In certain instances, in formulae AM-(I) and AM-(II), R¹ is—CH₂CH₂CH₂NH(C(═NH)(NH₂)) or —CH₂CH₂CH₂CH₂NH₂. In certain instances, informulae AM-(I) and AM-(II), R¹ is —CH₂CH₂CH₂NH(C(═NH)(NH₂)) or—CH₂CH₂CH₂CH₂NH₂, the configuration of the carbon atom to which R¹ isattached corresponding with that in an L-amino acid. In certaininstances, in formulae AM-(I) and AM-(II), R¹ is—CH₂CH₂CH₂NH(C(═NH)(NH₂)), the configuration of the carbon atom to whichR¹ is attached corresponding with that in an L-amino acid. In certaininstances, in formulae AM-(I) and AM-(II), R¹ is —CH₂CH₂CH₂CH₂NH₂, theconfiguration of the carbon atom to which R¹ is attached correspondingwith that in an L-amino acid.

In formulae AM-(I) and AM-(II), R² is an acyl, substituted acyl, or anN-acyl derivative of a peptide. In certain instances, R² is acyl. Incertain instances, R² is substituted acyl. In certain instances, R² isacetyl, benzoyl, malonyl, piperonyl or succinyl. In certain instances,R² is acetyl, malonyl, or succinyl. In certain instances, R² is acetyl.In certain instances, R² is malonyl. In certain instances, R² issuccinyl. In certain instances, R² is an N-acyl derivative of a peptide.

In certain instances, R² is a peptide of the formula (R⁴)_(p), wherein pis an integer from 1 to 100, and each R⁴ is an independently selectedamino acid, wherein the R⁴ at the terminal end of the peptide isN-acylated. In certain instances, each R⁴ is an independently selectedL-amino acid. In certain instances, p is an integer from 1 to 90, 80,70, 60, 50, 40, 30, 20, or 10. In certain instances, p is 1, 2, 3, 4, 5,6, 7, 8, 9, or 10. In certain instances, the terminal end of the peptideis N-acylated, wherein the acyl group is acetyl, benzoyl, malonyl,piperonyl or succinyl. In certain instances, the terminal end of thepeptide is N-acylated, wherein the acyl group is acetyl, malonyl, orsuccinyl. In certain instances, the terminal end of the peptide isN-acylated, wherein the acyl group is acetyl. In certain instances, theterminal end of the peptide is N-acylated, wherein the acyl group ismalonyl. In certain instances, the terminal end of the peptide isN-acylated, wherein the acyl group is succinyl.

The disclosure provides for a compound of the following formula:

or a salt, hydrate or solvate thereof.

The disclosure provides for a compound of the following formula:

or a salt, hydrate or solvate thereof.

The disclosure provides for a compound of the following formula:

or a salt, hydrate or solvate thereof.

In certain embodiments, in formulae AM-(I) and AM-(II),—C(O)—CH(R¹)—NHR² is a GI enzyme-cleavable moiety. A GI enzyme-cleavablemoiety is a structural moiety that is capable of being cleaved by a GIenzyme. In certain instances, a GI enzyme-cleavable moiety comprises acharged moiety that can fit into the active site of a GI enzyme and isable to orient the prodrug for cleavage at a scissile bond. Forinstance, the charged moiety can be a basic moiety that exists as acharged moiety at physiological pH.

For example, to form a GI enzyme-cleavable moiety, R¹ can include, butis not limited to, a side chain of lysine (such as L-lysine), a sidechain of arginine (such as L-arginine), a side chain of homolysine, aside chain of homoarginine, and a side chain of ornithine. Other GIenzyme-cleavable moieties include, but are not limited to, argininemimics, arginine homologues, arginine truncates, arginine with varyingoxidation states (for instance, metabolites), lysine mimics, lysinehomologues, lysine truncates, and lysine with varying oxidation states(for instance, metabolites). Examples of arginine and lysine mimicsinclude arylguanidines, arylamidines (substituted benzamidines),benzylamines and (bicyclo[2.2.2]octan-1-yl)methanamine and derivativesthereof.

In formulae AM-(I) and AM-(II), R² is selected from acyl, substitutedacyl, and N-acyl derivative of a peptide. In certain instances, informulae AM-(I) and AM-(II), R² is an N-acyl derivative of a peptide.The peptide may include one to 100 amino acids, where each amino acidcan be selected independently. In certain instances, there are one to 50amino acids in the peptide. In certain instances, there are one to 90,80, 70, 60, 50, 40, 30, 20, or 10 amino acids in the peptide. In certaininstances, there are about 100 amino acids in the peptide. In certaininstances, there are about 75 amino acids in the peptide. In certaininstances, there are about 50 amino acids in the peptide. In certaininstances, there are about 25 amino acids in the peptide. In certaininstances, there are about 20 amino acids in the peptide. In certaininstances, there are about 15 amino acids in the peptide. In certaininstances, there are about 10 amino acids in the peptide. In certaininstances, there are about 9 amino acids in the peptide. In certaininstances, there are about 8 amino acids in the peptide. In certaininstances, there are about 7 amino acids in the peptide. In certaininstances, there are about 6 amino acids in the peptide. In certaininstances, there are about 5 amino acids in the peptide. In certaininstances, there are about 4 amino acids in the peptide. In certaininstances, there are about 3 amino acids in the peptide. In certaininstances, there are about 2 amino acids in the peptide. In certaininstances, there is about 1 amino acid in the peptide.

In certain embodiments, in formulae AM-(I) and AM-(II),—C(O)—CH(R¹)—NHR² is a trypsin-cleavable moiety. A trypsin-cleavablemoiety is a structural moiety that is capable of being cleaved bytrypsin. In certain instances, a trypsin-cleavable moiety comprises acharged moiety that can fit into an active site of trypsin and is ableto orient the prodrug for cleavage at a scissile bond. For instance, thecharged moiety can be a basic moiety that exists as a charged moiety atphysiological pH.

In certain embodiments, in formulae AM-(I) and AM-(II), R¹ represents aside chain of an amino acid or a derivative of a side chain of an aminoacid that effects —C(O)—CH(R¹)—NHR² to be a trypsin-cleavable moiety. Aderivative refers to a substance that has been altered from anothersubstance by modification, partial substitution, homologation,truncation, or a change in oxidation state.

For example, to form a trypsin-cleavable moiety, R¹ can include, but isnot limited to, a side chain of lysine (such as L-lysine), arginine(such as L-arginine), homolysine, homoarginine, and ornithine. Othervalues for R¹ include, but are not limited to, arginine mimics, argininehomologues, arginine truncates, arginine with varying oxidation states(for instance, metabolites), lysine mimics, lysine homologues, lysinetruncates, and lysine with varying oxidation states (for instance,metabolites). Examples of arginine and lysine mimics includearylguanidines, arylamidines (substituted benzamidines), benzylaminesand (bicyclo[2.2.2]octan-1-yl)methanamine and derivatives thereof.

In certain instances, in formulae AM-(I) and AM-(II), R¹ represents—CH₂CH₂CH₂NH(C(═NH)(NH₂)) or —CH₂CH₂CH₂CH₂NH₂, the configuration of thecarbon atom to which R¹ is attached corresponding with that in anL-amino acid.

In formulae AM-(I) and AM-(II), R² is selected from acyl, substitutedacyl, and N-acyl derivative of a peptide. In certain instances, R² is anN-acyl derivative of an amino acid. In certain instances, R² is anN-acyl derivative of a peptide. The peptide may include one to 100 aminoacids and where each amino acid can be selected independently, and wherethe terminal amino acid is an N-acyl amino acid. In certain instances,there are one to 50 amino acids in the peptide. In certain instances,there are one to 90, 80, 70, 60, 50, 40, 30, 20, or 10 amino acids inthe peptide. In certain instances, there are about 100 amino acids inthe peptide. In certain instances, there are about 75 amino acids in thepeptide. In certain instances, there are about 50 amino acids in thepeptide. In certain instances, there are about 25 amino acids in thepeptide. In certain instances, there are about 20 amino acids in thepeptide. In certain instances, there are about 15 amino acids in thepeptide. In certain instances, there are about 10 amino acids in thepeptide. In certain instances, there are about 9 amino acids in thepeptide. In certain instances, there are about 8 amino acids in thepeptide. In certain instances, there are about 7 amino acids in thepeptide. In certain instances, there are about 6 amino acids in thepeptide. In certain instances, there are about 5 amino acids in thepeptide. In certain instances, there are about 4 amino acids in thepeptide. In certain instances, there are about 3 amino acids in thepeptide. In certain instances, there are about 2 amino acids in thepeptide. In certain instances, there is about 1 amino acid in thepeptide.

Formula AM-(III)

The present disclosure provides compounds of the general formulaAM-(III):

wherein

R is a GI enzyme-cleavable moiety or a trypsin-cleavable moiety.

Formula AM-(IV)

The present disclosure provides compounds of the general formulaAM-(IV):

wherein

R is a GI enzyme-cleavable moiety or a trypsin-cleavable moiety.

In certain embodiments, in formulae AM-(III) and AM-(IV), R is a GIenzyme-cleavable moiety. A GI enzyme-cleavable moiety is a structuralmoiety that is capable of being cleaved by GI enzyme. In certaininstances, a GI enzyme-cleavable moiety comprises a charged moiety thatcan fit into the active site of a GI enzyme and is able to orient theprodrug for cleavage at a scissile bond. For instance, the chargedmoiety can be a basic moiety that exists as a charged moiety atphysiological pH.

In certain embodiments, in formulae AM-(III) and AM-(IV), R is—C(O)—CH(R¹)—NH(R²), wherein R¹ represents a side chain of an amino acidor a derivative of a side chain of an amino acid that effects R to be aGI enzyme-cleavable moiety and R² is an acyl, substituted acyl, or anN-acyl derivative of a peptide. A derivative refers to a substance thathas been altered from another substance by modification, partialsubstitution, homologation, truncation, or a change in oxidation state.

For example, to form a GI enzyme-cleavable moiety, R¹ can include, butis not limited to, a side chain of lysine (such as L-lysine), arginine(such as L-arginine), homolysine, homoarginine, and ornithine. Othervalues for R include, but are not limited to, arginine mimics, argininehomologues, arginine truncates, arginine with varying oxidation states(for instance, metabolites), lysine mimics, lysine homologues, lysinetruncates, and lysine with varying oxidation states (for instance,metabolites). Examples of arginine and lysine mimics includearylguanidines, arylamidines (substituted benzamidines), benzylamines,and (bicyclo[2.2.2]octan-1-yl)methanamine and derivatives thereof.

In certain embodiments, in formulae AM-(III) and AM-(IV), R is atrypsin-cleavable moiety. A trypsin-cleavable moiety is a structuralmoiety that is capable of being cleaved by trypsin. In certaininstances, a trypsin-cleavable moiety comprises a charged moiety thatcan fit into an active site of trypsin and is able to orient the prodrugfor cleavage at a scissile bond. For instance, the charged moiety can bea basic moiety that exists as a charged moiety at physiological pH.

In certain embodiments, in formulae AM-(III) and AM-(IV), R is—C(O)—CH(R¹)—NH(R²), wherein R¹ represents a side chain of an amino acidor a derivative of a side chain of an amino acid that effects R to be atrypsin-cleavable moiety and R² is an acyl, substituted acyl, or anN-acyl derivative of a peptide. A derivative refers to a substance thathas been altered from another substance by modification, partialsubstitution, homologation, truncation, or a change in oxidation state.

For example, to form a trypsin-cleavable moiety, R¹ can include, but isnot limited to, a side chain of lysine (such as L-lysine), arginine(such as L-arginine), homolysine, homoarginine, and ornithine. Othervalues for R include, but are not limited to, arginine mimics, argininehomologues, arginine truncates, arginine with varying oxidation states(for instance, metabolites), lysine mimics, lysine homologues, lysinetruncates, and lysine with varying oxidation states (for instance,metabolites). Examples of arginine and lysine mimics includearylguanidines, arylamidines (substituted benzamidines), benzylaminesand (bicyclo[2.2.2]octan-1-yl)methanamine and derivatives thereof.

In certain instances, in formulae AM-(III) and AM-(IV), R¹ is—CH₂CH₂CH₂NH(C(═NH)(NH₂)) or —CH₂CH₂CH₂CH₂NH₂, the configuration of thecarbon atom to which R¹ is attached corresponding with that in anL-amino acid.

In certain embodiments, in formulae AM-(III) and AM-(IV), R² is acyl orsubstituted acyl. In certain instances, R² is an amino acid or an N-acylderivative of an amino acid.

In certain instances, R² is an N-acyl derivative of a peptide. In somecases, the peptide comprises one to 100 amino acids and where each aminoacid can be selected independently and the terminal amino acid isN-acylated. In certain instances, there are one to 50 amino acids in thepeptide. In certain instances, there are one to 90, 80, 70, 60, 50, 40,30, 20, or 10 amino acids in the peptide. In certain instances, thereare about 100 amino acids in the peptide. In certain instances, thereare about 75 amino acids in the peptide. In certain instances, there areabout 50 amino acids in the peptide. In certain instances, there areabout 25 amino acids in the peptide. In certain instances, there areabout 20 amino acids in the peptide. In certain instances, there areabout 15 amino acids in the peptide. In certain instances, there areabout 10 amino acids in the peptide. In certain instances, there areabout 9 amino acids in the peptide. In certain instances, there areabout 8 amino acids in the peptide. In certain instances, there areabout 7 amino acids in the peptide. In certain instances, there areabout 6 amino acids in the peptide. In certain instances, there areabout 5 amino acids in the peptide. In certain instances, there areabout 4 amino acids in the peptide. In certain instances, there areabout 3 amino acids in the peptide. In certain instances, there areabout 2 amino acids in the peptide. In certain instances, there is about1 amino acid in the peptide.

The present disclosure also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a compound of formulae AM-(I) or AM-(II) or apharmaceutically acceptable salt or solvate or stereoisomer thereof. Incertain embodiments, the pharmaceutical composition comprises a compoundof formula AM-(I). In certain embodiments, the pharmaceuticalcomposition comprises a compound of formula AM-(II). The presentdisclosure also provides pharmaceutical compositions comprising apharmaceutically acceptable carrier and a therapeutically effectiveamount of Compound AM-1, Compound AM-2, or Compound AM-5 or apharmaceutically acceptable salt or solvate or stereoisomer thereof. Incertain embodiments, the pharmaceutical composition comprises CompoundAM-1. In certain embodiments, the pharmaceutical composition comprisesCompound AM-2. In certain embodiments, the pharmaceutical compositioncomprises Compound AM-5.

The present disclosure also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a compound of formulae AM-(III) or AM-(IV) or apharmaceutically acceptable salt or solvate or stereoisomer thereof. Incertain embodiments, the pharmaceutical composition comprises a compoundof formula AM-(III). In certain embodiments, the pharmaceuticalcomposition comprises a compound of formula AM-(IV).

General Synthetic Procedures for Compounds AM-1 and AM-2

The compounds described herein may be obtained via the routesgenerically illustrated in Scheme 1.

The promoieties described herein, may be prepared and attached tocompounds containing amino groups by procedures known to those of skillin the art (See e.g., Green et al., “Protective Groups in OrganicChemistry,” (Wiley, 2^(nd) ed. 1991); Harrison et al., “Compendium ofSynthetic Organic Methods,” Vols. 1-8 (John Wiley and Sons, 1971-1996);“Beilstein Handbook of Organic Chemistry,” Beilstein Institute ofOrganic Chemistry, Frankfurt, Germany; Feiser et al., “Reagents forOrganic Synthesis,” Volumes 1-17, (Wiley Interscience); Trost et al.,“Comprehensive Organic Synthesis,” (Pergamon Press, 1991);“Theilheimer's Synthetic Methods of Organic Chemistry,” Volumes 1-45,(Karger, 1991); March, “Advanced Organic Chemistry,” (WileyInterscience), 1991; Larock “Comprehensive Organic Transformations,”(VCH Publishers, 1989); Paquette, “Encyclopedia of Reagents for OrganicSynthesis,” (John Wiley & Sons, 1995), Bodanzsky, “Principles of PeptideSynthesis,” (Springer Verlag, 1984); Bodanzsky, “Practice of PeptideSynthesis,” (Springer Verlag, 1984). Further, starting materials may beobtained from commercial sources or via well established syntheticprocedures, supra.

Compounds AM-1 and AM-2 may be obtained via the routes genericallyillustrated in Scheme 1.

In Scheme 1, Compound SM is coupled with Boc-Arg(Pbf)-OH to formCompound A. Standard peptide coupling reagents can be used for thereaction. Suitable peptide coupling reagents include, but are notlimited to, EDCI and HOBt, PyBroP and diisopropylethylamine, or HATU.Then, the Boc group of Compound A is removed to yield Compound B. TheBoc group can be removed with acidic conditions. Suitable reagents thatcan be used for the deprotection reaction include trifluoroacetic acidand hydrochloric acid.

With further reference to Scheme 1, a malonyl group is attached toCompound B via a reaction with mono-tert-butyl malonate to form CompoundC. Reaction between Compound B and mono-tert-butyl malonate can be aidedwith use of activation reagents, such as symmetric anhydrides,O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HBTU), dicyclohexylcarbodiimide (DCC) diisopropylcarbodiimide(DIC)/1-hydroxybenzotriazole (HOBt), andbenzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate(BOP).

Then, the Pbf group of Compound C is removed to yield Compound AM-2. ThePbf group and can be removed with acidic conditions. A suitable reagentthat can be used for the deprotection reaction is trifluoroacetic acid.

In another synthetic route to obtain Compound AM-1, Compound B isacetylated at the amino group to yield Compound D. Acetylation of aminogroups can be performed with acetic anhydride, acetic acid, or an acetylhalide.

Then, the Pbf group of Compound D is removed to yield Compound AM-1. ThePbf group and can be removed with acidic conditions. A suitable reagentthat can be used for the deprotection reaction is trifluoroacetic acid.

Examples of Amphetamine Prodrugs

Examples of certain amphetamine prodrugs are shown below. In FormulaeCC-(I) to CC-(IV), AA can represent a side chain of an amino acid. Aminoacids, including amino acid variants, are discussed in a section herein.

Formula CC-(I)

A certain example is a compound of Formula CC-(I):

wherein

X is an amphetamine, wherein a hydrogen atom of the amphetamine aminogroup is replaced by a covalent bond to —C(O)—C(AA)-NR^(cc1)R^(cc2);

R^(cc1) and R^(cc2) are independently selected from hydrogen, alkyl,substituted alkyl, acyl, substituted acyl, cycloalkyl, substitutedcycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl,substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl;and

AA is a side chain of an amino acid.

A certain formula of CC-(I) is shown below:

wherein

R^(cc1) and R^(cc2) are independently selected from hydrogen, alkyl,substituted alkyl, acyl, cycloalkyl, substituted cycloalkyl,heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, and substituted heteroarylalkyl; and

AA is a side chain of an amino acid.

Formula CC-(II)

A certain example is a compound of Formula CC-(II):

wherein

X is an amphetamine, wherein a hydrogen atom of the amphetamine aminogroup is replaced by a covalent bond to —C(O)—NR^(cc3)—C(AA)-C(O)—Z;

R^(cc3) is selected from hydrogen, alkyl, substituted alkyl, acyl,cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substitutedheterocycloalkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, andsubstituted heteroarylalkyl;

AA is a side chain of an amino acid; and

Z is selected from NH—R^(cc4), O—R^(cc4), OH and NH₂; and

R^(cc4) is selected from hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, acyl, arylalkyl and substituted arylalkyl.

A certain formula of CC-(II) is shown below:

wherein

R^(cc3) is selected from hydrogen, alkyl, substituted alkyl, acyl,cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substitutedheterocycloalkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, andsubstituted heteroarylalkyl;

AA is a side chain of an amino acid; and

Z is selected from NH—R^(cc4), O—R^(cc4), OH and NH₂; and

R^(cc4) is selected from hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, acyl, arylalkyl and substituted arylalkyl.

Formula CC-(III)

A certain example is a compound of Formula CC-(III):

wherein

X is an amphetamine, wherein a hydrogen atom of the amphetamine aminogroup is replaced by a covalent bond to —C(O)—O—NR^(cc5); and

R^(cc5) is selected from

A certain formula of CC-(III) is shown below:

wherein

R^(cc5) is selected from

Formula CC-(IV)

A certain example is a compound of Formula CC-(IV):

wherein

X is an amphetamine, wherein a hydrogen atom of the amphetamine aminogroup is replaced by a covalent bond to —C(O)—; and

Z is amidino or guanidine.

A certain formula of CC-(IV) is shown below:

wherein

Z is amidino or guanidine.

Amino Acids Found in Prodrugs

“Amino acid” means a building block of a polypeptide. As used herein,“amino acid” includes the 20 common naturally occurring L-amino acidsand all amino acids variants. In certain embodiments, an amino acid is acleavable substrate for a gastrointestinal enzyme.

“Naturally occurring amino acids” means the 20 common naturallyoccurring L-amino acids, that is, alanine, arginine, asparagine,aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine and valine.

“Amino acid variants” means an amino acid other than any of the 20common naturally occurring L-amino acids that is hydrolysable by aprotease in a manner similar to the ability of a protease to hydrolyze anaturally occurring L-amino acid. Amino acid variants, thus, includeamino acids or analogs of amino acids other than the 20naturally-occurring amino acids. Amino acid variants include syntheticamino acids. Amino acid variants also include amino acid derivatives. Aderivative refers to a substance that has been altered from anothersubstance by modification, partial substitution, homologation,truncation, or a change in oxidation state while retaining the abilityto be cleaved by a GI enzyme.

Certain examples of amino acid variants include, but are not limited to:2-aminoindane-2-carboxylic acid, 2-aminoisobutyric acid,4-amino-phenylalanine, 5-hydroxylysine, biphenylalanine, citrulline,cyclohexylalanine, cyclohexylglycine, diethylglycine, dipropylglycine,homoarginine, homocitrulline, homophenylalanine, homoproline,homoserine, homotyrosine, hydroxyproline, lanthionine, naphthylalanine,norleucine, ornithine, phenylalanine(4-fluoro), phenylalanine(4-nitro),phenylglycine, pipecolic acid, tert-butylalanine, tert-butylglycine,tert-leucine, tetrahydroisoquinoline-3-carboxylic acid, α-aminobutyricacid, γ-amino butyric acid, 2,3-diaminoproprionic acid,phenylalanine(2,3,4,5,6 pentafluoro), aminohexanoic acid and derivativesthereof.

Certain examples of amino acid variants include, but are not limited to,N-methyl amino acids. For example, N-methyl-alanine, N-methyl asparticacid, N-methyl-glutamic acid, N-methyl-glycine (sarcosine) are N-methylamino acids.

Certain examples of amino acid variants include, but are not limited to:dehydroalanine, ethionine, hypusine, lanthionine, pyrrolysine,α-aminoisobutyric acid, selenomethionine and derivatives thereof.

Certain examples of amino acid variants include, but are not limited to:(3,2-amino benzoic acid, 2-amino methyl benzoic acid,2-amino-3-guanidinopropionic acid, 2-amino-3-methoxy benzoic acid,2-amino-3-ureidopropionic acid, 3-amino benzoic acid, 4-amino benzoicacid, 4-amino methyl benzoic acid, 4-nitroanthranillic acid,5-acetamido-2-aminobenzoic acid, butanoic acid (HMB), glutathione,homocysteine, statine, taurine, β-alanine, 2-hydroxy-4-(methylthio),(3,4)-diamino benzoic acid, (3,5)-diamino benzoic acid and derivativesthereof.

Certain examples of amino acid variants include, but are not limited to:(2 amino ethyl) cysteine, 2-amino-3-ethyoxybutanoic acid, buthionine,cystathion, cysteic acid, ethionine, ethoxytheorine, methylserine,N-ε-ε-dimethyl-lysine, N-ω-nitro-arginine, saccharopine, isoserinederivatives thereof, and combinations thereof.

Certain examples of amino acid variants include, but are not limited to:l-carnitine, selenocysteine, l-sarcosine, l-lysinol, benzoic acid,citric acid, choline, EDTA or succinic acid and derivatives thereof.

Certain examples of amino acid variants are amino alcohols. Examples ofamino alcohols include, but are not limited to: alaninol, indano,norephedrine, asparaginol, aspartimol, glutamol, leucinol, methioninol,phenylalaninol, prolinol, tryptophanol, valinol, isoleucinol, argininol,serinol, tyrosinol, threoninol, cysteinol, lysinol, histidinol andderivatives thereof.

Enzyme Inhibitors

The enzyme capable of cleaving the enzymatically-cleavable moiety of anamphetamine prodrug or amphetamine analog prodrug can be a peptidase,also called a protease. In certain embodiments, the enzyme is an enzymelocated in the gastrointestinal (GI) tract, i.e., a gastrointestinalenzyme, or a GI enzyme. The enzyme can be a digestive enzyme such as agastric, intestinal, pancreatic or brush border enzyme or enzyme of GImicrobial flora, such as those involved in peptide hydrolysis. Examplesinclude a pepsin, such as pepsin A or pepsin B; a trypsin; achymotrypsin; an elastase; a carboxypeptidase, such as carboxypeptidaseA or carboxypeptidase B; an aminopeptidase (such as aminopeptidase N oraminopeptidase A; an endopeptidase; an exopeptidase; adipeptidylaminopeptidase such as dipeptidylaminopeptidase IV; adipeptidase; a tripeptidase; or an enteropeptidase. In certainembodiments, the enzyme is a cytoplasmic protease located on or in theGI brush border. In certain embodiments, the enzyme is trypsin.Accordingly, in certain embodiments, the corresponding composition isadministered orally to the patient.

The disclosure provides for a composition comprising a GI enzymeinhibitor. Such an inhibitor can inhibit at least one of any of the GIenzymes disclosed herein. An example of a GI enzyme inhibitor is aprotease inhibitor, such as a trypsin inhibitor.

As used herein, the term “GI enzyme inhibitor” refers to any agentcapable of inhibiting the action of a GI enzyme on a substrate. Theability of an agent to inhibit a GI enzyme can be measured using assayswell known in the art.

In certain embodiments, the GI enzyme capable of cleaving theenzymatically-cleavable moiety may be a protease—the promoietycomprising the enzymatically-cleavable moiety being linked to theamphetamine or prodrug through an amide (e.g., a peptide: —NHC(O)—)bond. The disclosure provides for inhibitors of proteases.

Proteases can be classified as exopeptidases or endopeptidases. Examplesof exopeptidases include aminopeptidase and carboxypeptidase (A, B, orY). Examples of endopeptidases include trypsin, chymotrypsin, elastase,pepsin, and papain. The disclosure provides for inhibitors ofexopeptidase and endopeptidase.

In some embodiments, the enzyme is a digestive enzyme of a protein. Thedisclosure provides for inhibitors of digestive enzymes. A gastric phaseinvolves stomach enzymes, such as pepsin. An intestinal phase involvesenzymes in the small intestine duodenum, such as trypsin, chymotrypsin,elastase, carboxypeptidase A, and carboxypeptidase B. An intestinalbrush border phase involves enzymes in the small intestinal brushborder, such as aminopeptidase N, aminopeptidase A, endopeptidases,dipeptidases, dipeptidylaminopeptidase, and dipeptidylaminopeptidase IV.An intestinal intracellular phase involves intracellular peptidases,such as dipeptidases (i.e., iminopeptidase) and aminopeptidase.

In certain embodiments, the enzyme inhibitor in the disclosedcompositions is a peptidase inhibitor or protease inhibitor. In certainembodiments, the enzyme is a digestive enzyme such as a gastric,pancreatic or brush border enzyme, such as those involved in peptidehydrolysis. Examples include pepsin, trypsin, chymotrypsin, colipase,elastase, aminopeptidase N, aminopeptidase A, dipeptidylaminopeptidaseIV, tripeptidase or enteropeptidase.

Proteases can be inhibited by naturally occurring peptide or proteininhibitors, or by small molecule naturally occurring or syntheticinhibitors. Examples of protein or peptide inhibitors that are proteaseinhibitors include, but are not limited to, α1-antitrypsin from humanplasma, aprotinin, trypsin inhibitor from soybean (SBTI), Bowman-BirkInhibitor from soybean (BBSI), trypsin inhibitor from egg white(ovomucoid), chromostatin, and potato-derived carboxypeptidaseinhibitor. Examples of small molecule irreversible inhibitors that areprotease inhibitors include, but are not limited to, TPCK(1-chloro-3-tosylamido-4-phenyl-2-butanone), TLCK(1-chloro-3-tosylamido-7-amino-2-heptone), and PMSF (phenylmethylsulfonyl floride). Examples of small molecule irreversible inhibitorsthat are protease inhibitors include, but are not limited tobenzamidine, apixaban, camostat, 3,4-dichloroisocoumarin,ε-aminocaprionic acid, amastatin, lysianadioic acid,1,10-phenanthroline, cysteamine, and bestatin. Other examples of smallmolecule inhibitors are Compound 101, Compound 102, Compound 103,Compound 104, Compound 105, Compound 106, Compound 107, Compound 108,Compound 109 and Compound 110, described in more detail below.

The following table shows examples of gastrointestinal (GI) proteases,examples of their corresponding substrates, and examples ofcorresponding inhibitors.

Table of Examples of GI Proteases and Corresponding Susbtrates andInhibitors GI Protease Substrates Inhibitors Trypsin R_(n−1)= Arg, Lys,TLCK, Benzamidine, positively Apixaban, Bowman Birk charged residuesChymotrypsin R_(n−1) = Phe, Tyr, ε-Aminocaprionic Trp, bulky TPCKhydrophobic Bowman-Birk residues Pepsin R_(n) = Leu, Phe, Pepstatin,PMSF Trp, Tyr Carboypeptidase B R_(n) = Arg, Lys Potato-derivedinhibitor, Lysianadioic acid Carboypeptidase A R_(n) not = Arg, LysPotato-derived inhibitor, 1,10-phenanthroline Elastase R_(n−1) = Ala,Gly, α1-antitrypsin, Ser, small neutral 3,4-dichlorocoumarin residuesAminopeptidase All free Bestatin, Amastatin N-terminal AATrypsin Inhibitors

As used herein, the term “trypsin inhibitor” refers to any agent capableof inhibiting the action of trypsin on a substrate. The term “trypsininhibitor” also encompasses salts of trypsin inhibitors. The ability ofan agent to inhibit trypsin can be measured using assays well known inthe art. For example, in a typical assay, one unit corresponds to theamount of inhibitor that reduces the trypsin activity by onebenzoyl-L-arginine ethyl ester unit (BAEE-U). One BAEE-U is the amountof enzyme that increases the absorbance at 253 nm by 0.001 per minute atpH 7.6 and 25° C. See, for example, K. Ozawa, M. Laskowski, 1966, J.Biol. Chem. 241, 3955 and Y. Birk, 1976, Meth. Enzymol. 45, 700. Incertain instances, a trypsin inhibitor can interact with an active siteof trypsin, such as the S1 pocket and the S3/4 pocket. The S1 pocket hasan aspartate residue which has affinity for positively charged moiety.The S3/4 pocket is a hydrophobic pocket. The disclosure provides forspecific trypsin inhibitors and non-specific serine protease inhibitors.

There are many trypsin inhibitors known in the art, both those specificto trypsin and those that inhibit trypsin and other proteases such aschymotrypsin. The disclosure provides for trypsin inhibitors that areproteins, peptides, and small molecules. The disclosure provides fortrypsin inhibitors that are irreversible inhibitors or reversibleinhibitors. The disclosure provides for trypsin inhibitors that arecompetitive inhibitors, non-competitive inhibitors, or uncompetitiveinhibitors. The disclosure provides for natural, synthetic orsemi-synthetic trypsin inhibitors.

Trypsin inhibitors can be derived from a variety of animal or vegetablesources: for example, soybean, corn, lima and other beans, squash,sunflower, bovine and other animal pancreas and lung, chicken and turkeyegg white, soy-based infant formula, and mammalian blood. Trypsininhibitors can also be of microbial origin: for example, antipain; see,for example, H. Umezawa, 1976, Meth. Enzymol. 45, 678.

In one embodiment, the trypsin inhibitor is derived from soybean.Trypsin inhibitors derived from soybean (Glycine max) are readilyavailable and are considered to be safe for human consumption. Theyinclude, but are not limited to, SBTI, which inhibits trypsin, andBowman-Birk inhibitor, which inhibits trypsin and chymotrypsin. Suchtrypsin inhibitors are available, for example from Sigma-Aldrich, St.Louis, Mo., USA.

A trypsin inhibitor can be an arginine mimic or lysine mimic, eithernatural or synthetic compound. In certain embodiments, the trypsininhibitor is an arginine mimic or a lysine mimic, wherein the argininemimic or lysine mimic is a synthetic compound. As used herein, anarginine mimic or lysine mimic can include a compound capable of bindingto the P¹ pocket of trypsin and/or interfering with trypsin active sitefunction. The arginine or lysine mimic can be a cleavable ornon-cleavable moiety.

Examples of trypsin inhibitors, which are arginine mimics and/or lysinemimics, include, but not limited to, arylguanidine, benzamidine,3,4-dichloroisocoumarin, diisopropylfluorophosphate, gabexate mesylate,and phenylmethanesulfonyl fluoride, or substituted versions or analogsthereof. In certain embodiments, trypsin inhibitors comprise acovalently modifiable group, such as a chloroketone moiety, an aldehydemoiety, or an epoxide moiety. Other examples of trypsin inhibitors areaprotinin, camostat and pentamidine.

Other examples of trypsin inhibitors include compounds of formula:

wherein:

Q¹ is selected from —O-Q⁴ or -Q⁴-COOH, where Q⁴ is C₁-C₄ alkyl;

Q² is N or CH; and

Q³ is aryl or substituted aryl.

Certain trypsin inhibitors include compounds of formula:

wherein:

Q⁵ is —C(O)—COOH or —NH-Q⁶-Q⁷-SO₂—C₆H₅, where

Q⁶ is —(CH₂)_(p)—COOH;

Q⁷ is —(CH₂)_(r)—C₆H5;

Q⁸ is NH;

n is an integer from zero to two;

o is zero or one;

p is an integer from one to three; and

r is an integer from one to three.

Other examples of trypsin inhibitors include compounds of formula:

wherein:

Q⁵ is —C(O)—COOH or —NH-Q⁶-Q⁷-SO₂—C₆H₅, where

Q⁶ is —(CH₂)_(p)—COOH;

Q⁷ is —(CH₂)_(r)—C₆H5; and

p is an integer from one to three; and

r is an integer from one to three.

Certain trypsin inhibitors include the following:

Compound 101

(S)-ethyl 4-(5-guanidino-2- (naphthalene-2-sulfonamido)pentanoyl)piperazine- 1-carboxylate Compound 102

(S)-ethyl 4-(5-guanidino-2-(2,4,6- triisopropylphcnylsulfonamido)pentanoyl)piperazinc-1-carboxylate Compound 103

(S)-ethyl 1-(5-guanidino-2- (naphthalene-2-sulfonamido)pentanoyl)piperidine- 4-carboxylate Compound 104

(S)-ethyl 1-(5-guanidino-2-(2,4,6- triisopropylphenylsulfonamido)pentanoyl)piperidine-4-carboxylate Compound 105

(S)-6-(4-(5-guanidino-2- (naphthalene-2-sulfonamido)pentanoyl)piperazin- 1-yl)-6-oxohexanoic acid Compound 106

4-aminobenzimidamide (also 4-aminobenzamidine) Compound 107

3-(4-carbamimidoylphenyl)-2- oxopropanoic acid Compound 108

(S)-5-(4- carbamimidoylbenzylamino)-5- oxo-4-((R)-4-phenyl-2-(phenylmethylsulfonamido)butana mido)pentanoic acid Compound 109

6-carbamimidoylnaphthalen-2-yl 4- (diaminomethyleneamino)benzoateCompound 110

4,4′-(pentane-1,5- diylbis(oxy))dibenzimidamide

In certain embodiments, the trypsin inhibitor is SBTI, BBSI, Compound101, Compound 106, Compound 108, Compound 109, or Compound 110. Incertain embodiments, the trypsin inhibitor is camostat. In certainembodiments, the trypsin inhibitor is Compound 109.

In certain embodiments, the trypsin inhibitor is a compound of formulaT-I:

wherein

A represents a group of the following formula:

-   -   R^(t9) and R^(t10) each represents independently a hydrogen atom        or a C₁₋₄ alkyl group,

R^(t8) represents a group selected from the following formulae:

wherein R^(t11), R^(t12) and R^(t13) each represents independently

(1) a hydrogen atom,

(2) a phenyl group,

(3) a C₁₋₄ alkyl group substituted by a phenyl group,

(4) a C₁₋₁₀ alkyl group,

(5) a C₁₋₁₀ alkoxyl group,

(6) a C₂₋₁₀ alkenyl group having 1 to 3 double bonds,

(7) a C₂₋₁₀ alkynyl group having 1 to 2 triple bonds,

(8) a group of formula: R^(t15)—C(O)XR^(t16),

-   -   wherein R^(t15) represents a single bond or a C₁₋₈ alkylene        group,    -   X represents an oxygen atom or an NH-group, and    -   R^(t16) represents a hydrogen atom, a C₁₋₄ alkyl group, a phenyl        group or a C₁₋₄ alkyl group substituted by a phenyl group, or

(9) a C₃₋₇ cycloalkyl group;

the structure

represents a 4-7 membered monocyclic hetero-ring containing 1 to 2nitrogen or oxygen atoms,

R^(t14) represents a hydrogen atom, a C₁₋₄ alkyl group substituted by aphenyl group or a group of formula: COOR^(t17), wherein R^(t17)represents a hydrogen atom, a C₁₋₄ alkyl group or a C₁₋₄ alkyl groupsubstituted by a phenyl group;

provided that R^(t11), R^(t12) and R^(t13) do not representsimultaneously hydrogen atoms;

or nontoxic salts, acid addition salts or hydrates thereof.

In certain embodiments, the trypsin inhibitor is a compound selectedfrom the following:

In certain embodiments, the trypsin inhibitor is a compound of formulaT-II:

wherein

X is NH;

n is zero or one; and

R^(t1) is selected from hydrogen, halogen, nitro, alkyl, substitutedalkyl, alkoxy, carboxyl, alkoxycarbonyl, acyl, aminoacyl, guanidine,amidino, carbamide, amino, substituted amino, hydroxyl, cyano and—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)—N—R^(n1)R^(n2), wherein each m isindependently an integer from zero to 2; and R^(n1) and R^(n2) areindependently selected from hydrogen and C₁₋₄ alkyl.

In certain embodiments, in formula T-II, R^(t1) is guanidino or amidino.

In certain embodiments, in formula T-II, R^(t1) is—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)—N—R^(n1)R^(n2), wherein m is one andR^(n1) and R^(n2) are methyl.

In certain embodiments, the trypsin inhibitor is a compound of formulaT-III:

wherein

X is NH;

n is zero or one;

L^(t1) is selected from —C(O)—O—; —O—C(O)—; —O—(CH₂)_(m)—O—;—OCH₂—Ar^(t2)—CH₂O—; —C(O)—NR^(t3)—; and —NR^(t3)—C(O)—;

R^(t3) is selected from hydrogen, C₁₋₆ alkyl, and substituted C₁₋₆alkyl;

Ar^(t1) and Ar^(t2) are each independently a substituted orunsubstituted aryl group;

m is an integer from 1 to 3; and

R^(t2) is selected from hydrogen, halogen, nitro, alkyl, substitutedalkyl, alkoxy, carboxyl, alkoxycarbonyl, acyl, aminoacyl, guanidino,amidino, carbamide, amino, substituted amino, hydroxyl, cyano and—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)—N—R^(n1)R^(n2), wherein each m isindependently an integer from zero to 2; and R^(n1) and R^(n2) areindependently selected from hydrogen and C₁₋₄ alkyl.

In certain embodiments, in formula T-III, R^(t2) is guanidino oramidino.

In certain embodiments, in formula T-III, R^(t2) is—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)—N—R^(n1)R^(n2) wherein m is one andR^(n1) and R^(n2) are methyl.

In certain embodiments, the trypsin inhibitor is a compound of formulaT-IV:

wherein

each X is NH;

each n is independently zero or one;

L^(t1) is selected from —C(O)—O—; —O—C(O)—; —O—(CH₂)_(m)—O—;—OCH₂—Ar^(t2)—CH₂O—; —C(O)—NR^(t3)—; and —NR^(t3)—C(O)—;

R^(t3) is selected from hydrogen, C₁₋₆ alkyl, and substituted C₁₋₆alkyl;

Ar^(t1) and Ar^(t2) are each independently a substituted orunsubstituted aryl group; and

m is an integer from 1 to 3.

In certain embodiments, in formula T-IV, Ar^(t1) or Ar^(t2) is phenyl.

In certain embodiments, in formula T-IV, Ar^(t1) or Ar^(t2) is naphthyl.

In certain embodiments, the trypsin inhibitor is Compound 109.

In certain embodiments, the trypsin inhibitor is

In certain embodiments, the trypsin inhibitor is Compound 110 or abis-arylamidine variant thereof; see, for example, J. D. Geratz, M.C.-F. Cheng and R. R. Tidwell (1976) J Med. Chem. 19, 634-639.

It will be appreciated that the pharmaceutical composition according tothe embodiments may further comprise one or more additional trypsininhibitors.

It is to be appreciated that the invention also includes inhibitors ofother enzymes involved in protein assimilation that can be used incombination with a prodrug disclosed herein comprising an amino acid ofalanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, orvaline or amino acid variants thereof.

Combinations of Amphetamine Prodrugs and Enzyme Inhibitors

As discussed above, the present disclosure provides pharmaceuticalcompositions which comprise an enzyme inhibitor and an amphetamineprodrug that contains an enzyme-cleavable moiety that, when cleaved,facilitates release of amphetamine.

Examples of compositions containing an amphetamine prodrug and an enzymeinhibitor (e.g., a trypsin inhibitor) are described below.

Combinations of Formulae AM-(I) to AM-(II) and Enzyme Inhibitor

The embodiments provide a pharmaceutical composition, which comprises anenzyme inhibitor and a compound of general Formulae AM-(I) to AM-(IV),or a pharmaceutically acceptable salt thereof.

The embodiments provide a pharmaceutical composition, which comprises acompound of Formulae T-I to T-IV and a compound of general FormulaeAM-(I) to AM-(IV), or a pharmaceutically acceptable salt thereof. Theembodiments provide a pharmaceutical composition, which comprisesCompound 109 and a compound of general Formulae AM-(I) to AM-(IV), or apharmaceutically acceptable salt thereof.

Certain embodiments provide for a combination of a compound of FormulaAM-(I) and an enzyme inhibitor. In certain embodiments, combinations ofa compound of Formula AM-(I) and an enzyme inhibitor include, but arenot limited to the following: a compound of Formula AM-(I) and SBTI; acompound of Formula AM-(I) and BBSI; a compound of Formula AM-(I) andCompound 101; a compound of Formula AM-(I) and Compound 106; a compoundof Formula AM-(I) and Compound 108; a compound of Formula AM-(I) andCompound 109; a compound of Formula AM-(I) and Compound 110; or apharmaceutically acceptable salt thereof; and the like.

Certain embodiments provide for a combination of a compound of FormulaAM-(II) and an enzyme inhibitor. In certain embodiments, combinations ofa compound of Formula AM-(II) and an enzyme inhibitor include, but arenot limited to the following: a compound of Formula AM-(II) and SBTI; acompound of Formula AM-(II) and BBSI; a compound of Formula AM-(II) andCompound 101; a compound of Formula AM-(II) and Compound 106; a compoundof Formula AM-(II) and Compound 108; a compound of Formula AM-(II) andCompound 109; a compound of Formula AM-(II) and Compound 110; or apharmaceutically acceptable salt thereof; and the like.

Certain embodiments provide for a combination of a compound of FormulaAM-(III) and an enzyme inhibitor. In certain embodiments, combinationsof a compound of Formula AM-(III) and an enzyme inhibitor include, butare not limited to the following: a compound of Formula AM-(III) andSBTI; a compound of Formula AM-(III) and BBSI; a compound of FormulaAM-(III) and Compound 101; a compound of Formula AM-(III) and Compound106; a compound of Formula AM-(III) and Compound 108; a compound ofFormula AM-(III) and Compound 109; a compound of Formula AM-(III) andCompound 110; or a pharmaceutically acceptable salt thereof; and thelike.

Certain embodiments provide for a combination of a compound of FormulaAM-(IV) and an enzyme inhibitor. In certain embodiments, combinations ofa compound of Formula AM-(IV) and an enzyme inhibitor include, but arenot limited to the following: a compound of Formula AM-(IV) and SBTI; acompound of Formula AM-(IV) and BBSI; a compound of Formula AM-(IV) andCompound 101; a compound of Formula AM-(IV) and Compound 106; a compoundof Formula AM-(IV) and Compound 108; a compound of Formula AM-(IV) andCompound 109; a compound of Formula AM-(IV) and Compound 110; or apharmaceutically acceptable salt thereof; and the like.

Certain embodiments provide for a combination of Compound AM-1 and atrypsin inhibitor. Compound AM-1 is 2-Acetylamino-5-guanidino-pentanoicacid ((R)-1-methyl-2-phenylethyl)-amide, oramphetamine-arginine-acetate. In certain embodiments, combinations ofCompound AM-1 and a trypsin inhibitor include, but are not limited tothe following: Compound AM-1 and SBTI; Compound AM-1 and BBSI; CompoundAM-1 and Compound 101; Compound AM-1 and Compound 106; Compound AM-1 andCompound 108; Compound AM-1 and Compound 109; Compound AM-1 and Compound110; or a pharmaceutically acceptable salt thereof; and the like.

Certain embodiments provide for a combination of Compound AM-2 and atrypsin inhibitor. Compound AM-2 isN-[4-Guanidino-1-((R)-1-methyl-2-phenyl-ethylcarbamoyl)-butyl]-malonicacid, or amphetamine-arginine-malonic acid. In certain embodiments,combinations of Compound AM-2 and a trypsin inhibitor include, but arenot limited to the following: Compound AM-2 and SBTI; Compound AM-2 andBBSI; Compound AM-2 and Compound 101; Compound AM-2 and Compound 106;Compound AM-2 and Compound 108; Compound AM-2 and Compound 109; CompoundAM-2 and Compound 110; or a pharmaceutically acceptable salt thereof;and the like.

Certain embodiments provide for a combination of Compound AM-5 and atrypsin inhibitor. Compound AM-5 is amphetamine-lysine-acetate. Incertain embodiments, combinations of Compound AM-5 and a trypsininhibitor include, but are not limited to the following: Compound AM-5and SBTI; Compound AM-5 and BBSI; Compound AM-5 and Compound 101;Compound AM-5 and Compound 106; Compound AM-2 and Compound 108; CompoundAM-5 and Compound 109; Compound AM-5 and Compound 110; or apharmaceutically acceptable salt thereof; and the like.

The embodiments provide a pharmaceutical composition, which comprisesCompound 109 and a compound of formula AM-(I) or a pharmaceuticallyacceptable salt thereof. The embodiments provide a pharmaceuticalcomposition, which comprises Compound 109 and a compound of formulaAM-(II) or a pharmaceutically acceptable salt thereof. The embodimentsprovide a pharmaceutical composition, which comprises Compound 109 andCompound AM-1 or a pharmaceutically acceptable salt thereof. Theembodiments provide a pharmaceutical composition, which comprisesCompound 109 and Compound AM-2 or a pharmaceutically acceptable saltthereof. The embodiments provide a pharmaceutical composition, whichcomprises Compound 109 and Compound AM-5 or a pharmaceuticallyacceptable salt thereof

Combinations of Amphetamine Prodrugs and Other Drugs

The disclosure provides for an amphetamine prodrug and a further prodrugor drug included in a pharmaceutical composition. Such a prodrug or drugmay provide additional stimulant effects or may have effects other than,or in addition to, the effects associated with amphetamines. Embodimentsprovide a pharmaceutical composition, which comprises an amphetamineprodrug and a further prodrug or drug and optionally comprises an enzymeinhibitor. Also included are pharmaceutically acceptable salts thereof.

In certain embodiments, the enzyme inhibitor is selected from SBTI,BBSI, Compound 101, Compound 106, Compound 108, Compound 109, andCompound 110. In certain embodiments, the enzyme inhibitor is camostat.

In certain embodiments, a pharmaceutical composition can comprise anamphetamine prodrug, a non-amphetamine drug and at least one enzymeinhibitor.

Pharmaceutical Compositions and Methods of Use

The pharmaceutical composition according to the embodiments can furthercomprise a pharmaceutically acceptable carrier. The composition isconveniently formulated in a form suitable for oral (including buccaland sublingual) administration, for example as a tablet, capsule, thinfilm, powder, suspension, solution, syrup, dispersion or emulsion. Thecomposition can contain components conventional in pharmaceuticalpreparations, e.g., one or more carriers, binders, lubricants,excipients (e.g., to impart controlled release characteristics), pHmodifiers, sweeteners, bulking agents, coloring agents or further activeagents.

Patients can be humans, and also other mammals, such as livestock, zooanimals and companion animals, such as a cat, dog or horse.

In some aspects, the embodiments provide a pharmaceutical composition asdescribed herein for use in the treatment of conditions such as, but notlimited to, Attention Deficit Hyperactivity Disorder (ADHD), ChronicFatigue Syndrome (CFS), brain injuries, narcolepsy, obesity, etc. Thepharmaceutical composition according to the embodiments is useful, forexample, in the treatment of a patient suffering from, ADHD, CFS, braininjury, narcolepsy, obesity, etc. Accordingly, the present disclosureprovides methods of treating or preventing ADHD, CFS, brain injury,narcolepsy, or obesity in a subject, the methods involving administeringto the subject a disclosed composition. The present disclosure providesfor a disclosed composition for use in therapy or prevention or as amedicament. The present disclosure also provides the use of a disclosedcomposition for the manufacture of a medicament, especially for themanufacture of a medicament for the treatment or prevention of ADHD,CFS, brain injury, narcolepsy, or obesity.

The present disclosure provides use of an amphetamine prodrug and anenzyme inhibitor, such as a trypsin inhibitor, in the treatment of ADHD,CFS, brain injury, narcolepsy, or obesity.

The present disclosure provides use of an amphetamine prodrug and anenzyme inhibitor, such as a trypsin inhibitor, in the prevention ofADHD, CFS, brain injury, narcolepsy, or obesity.

The present disclosure provides use of an amphetamine prodrug and anenzyme inhibitor, such as a trypsin inhibitor, in the manufacture of amedicament for treatment of ADHD, CFS, brain injury, narcolepsy, orobesity. The present disclosure provides use of an amphetamine prodrugand an enzyme inhibitor, such as a trypsin inhibitor, in the manufactureof a medicament for prevention of ADHD, CFS, brain injury, narcolepsy,or obesity.

In another aspect, the embodiments provide a method of treating ADHD,CFS, brain injury, narcolepsy, or obesity in a patient requiringtreatment, which comprises administering an effective amount of apharmaceutical composition as described herein. In another aspect, theembodiments provides a method of preventing ADHD, CFS, brain injury,narcolepsy, or obesity in a patient requiring treatment, which comprisesadministering an effective amount of a pharmaceutical composition asdescribed herein.

The amount of composition disclosed herein to be administered to apatient to be effective (i.e., to provide blood levels of amphetaminesufficient to be effective in the treatment or prophylaxis of ADHD, CFS,brain injury, narcolepsy, or obesity) will depend upon thebioavailability of the particular composition, the susceptibility of theparticular composition to enzyme activation in the gut, the amount andpotency of enzyme inhibitor (e.g., trypsin inhibitor) present in thecomposition, as well as other factors, such as the species, age, weight,sex, and condition of the patient, manner of administration and judgmentof the prescribing physician. In general, the composition dose can besuch that the amphetamine prodrug is in the range of from 0.01milligrams prodrug per kilogram to 20 milligrams prodrug per kilogram(mg/kg) body weight. For example, a composition comprising a residue ofamphetamine can be administered at a dose equivalent to administeringfree amphetamine in the range of from 0.01 mg/kg to 40 mg/kg bodyweight, or 0.1 to 30 mg/kg body weight, or 0.2 to 20 mg/kg body weight.In one embodiment wherein the composition comprises an amphetamineprodrug, the composition can be administered at a dose such that thelevel of amphetamine achieved in the blood is in the range of from 0.5ng/ml to 200 ng/ml.

The amount of an enzyme inhibitor (e.g., a trypsin inhibitor) to beadministered to the patient to be effective (i.e., to attenuate releaseof amphetamine when administration of an amphetamine prodrug disclosedherein alone would lead to overexposure of amphetamine) will depend uponthe effective dose of the particular prodrug and the potency of theparticular enzyme inhibitor, as well as other factors, such as thespecies, age, weight, sex and condition of the patient, manner ofadministration and judgment of the prescribing physician. In general,the dose of enzyme inhibitor can be in the range of from 0.001 mg to 50mg per mg of prodrug disclosed herein. In a certain embodiment, the doseof enzyme inhibitor can be in the range of from 0.05 mg to 50 mg per mgof prodrug disclosed herein. In one embodiment, the dose of enzymeinhibitor can be in the range of from 0.01 nanomoles to 100 micromolesper micromole of prodrug disclosed herein.

Representative Embodiments of Dose Units of Prodrug and GI EnzymeInhibitor Having a Desired Pharmacokinetic Profile

The embodiments include a composition that comprises (a) an amphetamineprodrug of Formulae AM-(I) or AM-(II), which comprises amphetaminecovalently bound to a promoiety comprising a GI enzyme-cleavable moiety,wherein cleavage of the GI enzyme-cleavable moiety by a GI enzymemediates release of the amphetamine, and (b) a GI enzyme inhibitor thatinteracts with the GI enzyme that mediates enzymatically-controlledrelease of the amphetamine from the prodrug following ingestion of thecomposition. In one embodiment, the GI enzyme is trypsin, the GIenzyme-cleavable moiety is a trypsin-cleavable moiety, and the GI enzymeinhibitor is a trypsin inhibitor.

The embodiments include a dose unit comprising a composition, such as apharmaceutical composition, comprising an amphetamine prodrug ofFormulae AM-(I) or AM-(II) and a GI enzyme inhibitor, where theamphetamine prodrug of Formulae AM-(I) or AM-(II) and GI enzymeinhibitor are present in the dose unit in an amount effective to providefor a pre-selected pharmacokinetic (PK) profile following ingestion. Infurther embodiments, the pre-selected PK profile comprises at least onePK parameter value that is less than the PK parameter value ofamphetamine released following ingestion of an equivalent dosage of anamphetamine prodrug of Formulae AM-(I) or AM-(II) in the absence ofinhibitor. In further embodiments, the PK parameter value is selectedfrom an amphetamine Cmax value, an amphetamine exposure value, and a(1/amphetamine Tmax) value.

In certain embodiments, the dose unit provides for a pre-selected PKprofile following ingestion of at least two dose units. In relatedembodiments, the pre-selected PK profile of such dose units is modifiedrelative to the PK profile following ingestion of an equivalent dosageof an amphetamine prodrug of Formulae AM-(I) or AM-(II) withoutinhibitor. In related embodiments, such a dose unit provides thatingestion of an increasing number of the dose units provides for alinear PK profile. In related embodiments, such a dose unit providesthat ingestion of an increasing number of the dose units provides for anonlinear PK profile. In related embodiments, the PK parameter value ofthe PK profile of such a dose unit is selected from an amphetamine Cmaxvalue, a (1/amphetamine Tmax) value, and an amphetamine exposure value.

The embodiments include methods for treating a patient comprisingadministering any of the compositions, such as pharmaceuticalcompositions, comprising an amphetamine prodrug of Formulae AM-(I) orAM-(II) and a GI enzyme inhibitor or dose units described herein to apatient in need thereof. The embodiments include methods to reduce sideeffects of a therapy comprising administering any of such compositions,e.g., pharmaceutical compositions, or dose units described herein, to apatient in need thereof. The embodiments include methods of improvingpatient compliance with a therapy prescribed by a clinician comprisingdirecting administration of any of such compositions, e.g.,pharmaceutical compositions, or dose units described herein, to apatient in need thereof. Such embodiments can provide for improvedpatient compliance with a prescribed therapy as compared to patientcompliance with a prescribed therapy using drug and/or using prodrugwithout inhibitor as compared to prodrug with inhibitor.

The embodiments include methods of reducing risk of unintended overdoseof amphetamine comprising directing administration of any of suchcompositions, e.g., pharmaceutical compositions, or dose units describedherein, to a patient in need of treatment.

The embodiments include methods of making a dose unit comprisingcombining an amphetamine prodrug of Formulae AM-(I) or AM-(II) and a GIenzyme inhibitor in a dose unit, wherein the amphetamine prodrug ofFormulae AM-(I) or AM-(II) and GI enzyme inhibitor are present in thedose unit in an amount effective to attenuate release of amphetaminefrom the amphetamine prodrug of Formulae AM-(I) or AM-(II).

The embodiments include methods of deterring misuse or abuse of multipledose units of an amphetamine prodrug of Formulae AM-(I) or AM-(II)comprising combining an amphetamine prodrug of Formulae AM-(I) orAM-(II) and a GI enzyme inhibitor in a dose unit, wherein theamphetamine prodrug of Formulae AM-(I) or AM-(II) and GI enzymeinhibitor are present in the dose unit in an amount effective toattenuate release of amphetamine from the amphetamine prodrug ofFormulae AM-(I) or AM-(II) such that ingestion of multiples of doseunits by a patient does not provide a proportional release of theamphetamine. In further embodiments, release of drug is decreasedcompared to release of drug by an equivalent dosage of prodrug in theabsence of inhibitor.

One embodiment is a method for identifying a GI enzyme inhibitor andprodrug of Formulae AM-(I) or AM-(II) suitable for formulation in a doseunit. Such a method can be conducted as, for example, an in vitro assay,an in vivo assay, or an ex vivo assay. In one embodiment, the GI enzymeinhibitor is a trypsin inhibitor.

The embodiments include methods for identifying a GI enzyme inhibitorand prodrug of Formulae AM-(I) or AM-(II) suitable for formulation in adose unit comprising combining a prodrug of Formulae AM-(I) or AM-(II),a GI enzyme inhibitor, and a GI enzyme in a reaction mixture, anddetecting prodrug conversion, wherein a decrease in prodrug conversionin the presence of the GI enzyme inhibitor as compared to prodrugconversion in the absence of the GI enzyme inhibitor indicates the GIenzyme inhibitor and prodrug of Formulae AM-(I) or AM-(II) are suitablefor formulation in a dose unit.

The embodiments include methods for identifying a GI enzyme inhibitorand prodrug of Formulae AM-(I) or AM-(II) suitable for formulation in adose unit comprising administering to an animal a GI enzyme inhibitorand prodrug of Formulae AM-(I) or AM-(II) and detecting prodrugconversion, wherein a decrease in amphetamine conversion in the presenceof the GI enzyme inhibitor as compared to amphetamine conversion in theabsence of the GI enzyme inhibitor indicates the GI enzyme inhibitor andprodrug of Formulae AM-(I) or AM-(II) are suitable for formulation in adose unit. In certain embodiments, administering comprises administeringto the animal increasing doses of inhibitor co-dosed with a selectedfixed dose of prodrug. Detecting prodrug conversion can facilitateidentification of a dose of inhibitor and a dose of prodrug thatprovides for a pre-selected pharmacokinetic (PK) profile. Such methodscan be conducted as, for example, an in vivo assay or an ex vivo assay.

The embodiments include methods for identifying a GI enzyme inhibitorand prodrug of Formulae I-XII suitable for formulation in a dose unitcomprising administering to an animal tissue a GI enzyme inhibitor andprodrug of Formulae AM-(I) or AM-(II) and detecting prodrug conversion,wherein a decrease in prodrug conversion in the presence of the GIenzyme inhibitor as compared to prodrug conversion in the absence of theGI enzyme inhibitor indicates the GI enzyme inhibitor and prodrug ofFormulae AM-(I) or AM-(II) are suitable for formulation in a dose unit.

Dose Units of Prodrug and Inhibitor Having a Desired PharmacokineticProfile

The present disclosure provides dose units of prodrug and inhibitor thatcan provide for a desired pharmacokinetic (PK) profile. Dose units canprovide a modified PK profile compared to a reference PK profile asdisclosed herein. It will be appreciated that a modified PK profile canprovide for a modified pharmacodynamic (PD) profile. Ingestion ofmultiples of such a dose unit can also provide a desired PK profile.

Unless specifically stated otherwise, “dose unit” as used herein refersto a combination of a GI enzyme-cleavable prodrug (e.g., atrypsin-cleavable prodrug) and a GI enzyme inhibitor (e.g., a trypsininhibitor). A “single dose unit” is a single unit of a combination of aGI enzyme-cleavable prodrug (e.g., a trypsin-cleavable prodrug) and a GIenzyme inhibitor (e.g., a trypsin inhibitor), where the single dose unitprovide a therapeutically effective amount of drug (i.e., a sufficientamount of drug to produce a therapeutic effect, e.g., a dose within therespective drug's therapeutic window, or therapeutic range). “Multipledose units” or “multiples of a dose unit” or a “multiple of a dose unit”refers to at least two single dose units.

As used herein, a “PK profile” refers to a profile of drug concentrationin blood or plasma. Such a profile can be a relationship of drugconcentration over time (i.e., a “concentration-time PK profile”) or arelationship of drug concentration versus number of doses ingested(i.e., a “concentration-dose PK profile”.) A PK profile is characterizedby PK parameters.

As used herein, a “PK parameter” refers to a measure of drugconcentration in blood or plasma, such as: (1) “drug Cmax”, the maximumconcentration of drug achieved in blood or plasma; (2) “drug Tmax”, thetime elapsed following ingestion to achieve Cmax; and (3) “drugexposure”, the total concentration of drug present in blood or plasmaover a selected period of time, which can be measured using the areaunder the curve (AUC) of a time course of drug release over a selectedperiod of time (t). Modification of one or more PK parameters providesfor a modified PK profile.

For purposes of describing the features of dose units of the presentdisclosure, “PK parameter values” that define a PK profile include drugCmax (e.g., amphetamine Cmax), total drug exposure (e.g., area under thecurve) (e.g., amphetamine exposure) and 1/(drug Tmax) (such that adecreased 1/Tmax is indicative of a delay in Tmax relative to areference Tmax) (e.g., 1/amphetamine Tmax). Thus, a decrease in a PKparameter value relative to a reference PK parameter value can indicate,for example, a decrease in drug Cmax, a decrease in drug exposure,and/or a delayed Tmax.

Dose units of the present disclosure can be adapted to provide for amodified PK profile, e.g., a PK profile that is different from thatachieved from dosing a given dose of prodrug in the absence of inhibitor(i.e., without inhibitor). For example, dose units can provide for atleast one of decreased drug Cmax, delayed drug Tmax and/or decreaseddrug exposure compared to ingestion of a dose of prodrug in the sameamount but in the absence of inhibitor. Such a modification is due tothe inclusion of an inhibitor in the dose unit.

As used herein, “a pharmacodynamic (PD) profile” refers to a profile ofthe efficacy of a drug in a patient (or subject or user), which ischaracterized by PD parameters. “PD parameters” include “drug Emax” (themaximum drug efficacy), “drug EC50” (the concentration of drug at 50% ofthe Emax), and side effects.

FIG. 1 is a schematic illustrating an example of the effect ofincreasing inhibitor concentrations upon the PK parameter drug Cmax fora fixed dose of prodrug. At low concentrations of inhibitor, there maybe no detectable effect on drug release, as illustrated by the plateauportion of the plot of drug Cmax (Y-axis) versus inhibitor concentration(X-axis). As inhibitor concentration increases, a concentration isreached at which drug release from prodrug is attenuated, causing adecrease in, or suppression of, drug Cmax. Thus, the effect of inhibitorupon a prodrug PK parameter for a dose unit of the present disclosurecan range from undetectable, to moderate, to complete inhibition (i.e.,no detectable drug release).

A dose unit can be adapted to provide for a desired PK profile (e.g., aconcentration-time PK profile) following ingestion of a single dose. Adose unit can be adapted to provide for a desired PK profile (e.g., aconcentration-dose PK profile) following ingestion of multiple doseunits (e.g., at least 2, at least 3, at least 4 or more dose units).

Dose Units Providing Modified PK Profiles

A combination of a prodrug and an inhibitor in a dose unit can provide adesired (or “pre-selected”) PK profile (e.g., a concentration-time PKprofile) following ingestion of a single dose.

The PK profile of such a dose unit can be characterized by one or moreof a pre-selected drug Cmax, a pre-selected drug Tmax or a pre-selecteddrug exposure. The PK profile of the dose unit can be modified comparedto a PK profile achieved from the equivalent dosage of prodrug in theabsence of inhibitor (i.e., a dose that is the same as the dose unitexcept that it lacks inhibitor).

A modified PK profile can have a decreased PK parameter value relativeto a reference PK parameter value (e.g., a PK parameter value of a PKprofile following ingestion of a dosage of prodrug that is equivalent toa dose unit except without inhibitor). For example, a dose unit canprovide for a decreased drug Cmax, decreased drug exposure, and/ordelayed drug Tmax.

FIG. 2 presents schematic graphs showing examples of modifiedconcentration-time PK profiles of a single dose unit. Panel A is aschematic of drug concentration in blood or plasma (Y-axis) following aperiod of time (X-axis) after ingestion of prodrug in the absence orpresence of inhibitor. The solid, top line in Panel A provides anexample of drug concentration following ingestion of prodrug withoutinhibitor. The dashed, lower line in Panel A represents drugconcentration following ingestion of the same dose of prodrug withinhibitor. Ingestion of inhibitor with prodrug provides for a decreaseddrug Cmax relative to the drug Cmax that results from ingestion of thesame amount of prodrug in the absence of inhibitor. Panel A alsoillustrates that the total drug exposure following ingestion of prodrugwith inhibitor is also decreased relative to ingestion of the sameamount of prodrug without inhibitor.

Panel B of FIG. 2 provides another example of a dose unit having amodified concentration-time PK profile. As in Panel A, the solid topline represents drug concentration over time in blood or plasmafollowing ingestion of prodrug without inhibitor, while the dashed lowerline represents drug concentration following ingestion of the sameamount of prodrug with inhibitor. In this example, the dose unitprovides a PK profile having a decreased drug Cmax, decreased drugexposure, and a delayed drug Tmax (i.e., decreased (1/drug Tmax)relative to ingestion of the same dose of prodrug without inhibitor.

Panel C of FIG. 2 provides another example of a dose unit having amodified concentration-time PK profile. As in Panel A, the solid linerepresents drug concentration over time in blood or plasma followingingestion of prodrug without inhibitor, while the dashed line representsdrug concentration following ingestion of the same amount of prodrugwith inhibitor. In this example, the dose unit provides a PK profilehaving a delayed drug Tmax (i.e., decreased (1/drug Tmax) relative toingestion of the same dose of prodrug without inhibitor.

Dose units that provide for a modified PK profile (e.g., a decreaseddrug Cmax and/or delayed drug Tmax as compared to, a PK profile of drugor a PK profile of prodrug without inhibitor), find use in tailoring ofdrug dose according to a patient's needs (e.g., through selection of aparticular dose unit and/or selection of a dosage regimen), reduction ofside effects, and/or improvement in patient compliance (as compared toside effects or patient compliance associated with drug or with prodrugwithout inhibitor). As used herein, “patient compliance” refers towhether a patient follows the direction of a clinician (e.g., aphysician) including ingestion of a dose that is neither significantlyabove nor significantly below that prescribed. Such dose units alsoreduce the risk of misuse, abuse or overdose by a patient as compared tosuch risk(s) associated with drug or prodrug without inhibitor. Forexample, dose units with a decreased drug Cmax provide less reward foringestion than does a dose of the same amount of drug, and/or the sameamount of prodrug without inhibitor.

Dose Units Providing Modified PK Profiles Upon Ingestion of MultipleDose Units

A dose unit of the present disclosure can be adapted to provide for adesired PK profile (e.g., a concentration-time PK profile orconcentration-dose PK profile) following ingestion of multiples of adose unit (e.g., at least 2, at least 3, at least 4, or more doseunits). A concentration-dose PK profile refers to the relationshipbetween a selected PK parameter and a number of single dose unitsingested. Such a profile can be dose proportional, linear (a linear PKprofile) or nonlinear (a nonlinear PK profile). A modifiedconcentration-dose PK profile can be provided by adjusting the relativeamounts of prodrug and inhibitor contained in a single dose unit and/orby using a different prodrug and/or inhibitor.

FIG. 3 provides schematics of examples of concentration-dose PK profiles(exemplified by drug Cmax, Y-axis) that can be provided by ingestion ofmultiples of a dose unit (X-axis) of the present disclosure. Eachprofile can be compared to a concentration-dose PK profile provided byincreasing doses of drug alone, where the amount of drug in the blood orplasma from one dose represents a therapeutically effective amountequivalent to the amount of drug released into the blood or plasma byone dose unit of the disclosure. Such a “drug alone” PK profile istypically dose proportional, having a forty-five degree angle positivelinear slope. It is also to be appreciated that a concentration-dose PKprofile resulting from ingestion of multiples of a dose unit of thedisclosure can also be compared to other references, such as aconcentration-dose PK profile provided by ingestion of an increasingnumber of doses of prodrug without inhibitor wherein the amount of drugreleased into the blood or plasma by a single dose of prodrug in theabsence of inhibitor represents a therapeutically effective amountequivalent to the amount of drug released into the blood or plasma byone dose unit of the disclosure.

As illustrated by the relationship between prodrug and inhibitorconcentration in FIG. 1, a dose unit can include inhibitor in an amountthat does not detectably affect drug release following ingestion.Ingestion of multiples of such a dose unit can provide aconcentration-dose PK profile such that the relationship between numberof dose units ingested and PK parameter value is linear with a positiveslope, which is similar to, for example, a dose proportional PK profileof increasing amounts of prodrug alone. Panel A of FIG. 3 depicts such aprofile. Dose units that provide a concentration-dose PK profile havingsuch an undetectable change in drug Cmax in vivo compared to the profileof prodrug alone can find use in thwarting enzyme conversion of prodrugfrom a dose unit that has sufficient inhibitor to reduce or prevent invitro cleavage of the enzyme-cleavable prodrug by its respective enzyme.

Panel B in FIG. 3 represents a concentration-dose PK profile such thatthe relationship between the number of dose units ingested and a PKparameter value is linear with positive slope, where the profileexhibits a reduced slope relative to panel A. Such a dose unit providesa profile having a decreased PK parameter value (e.g., drug Cmax)relative to a reference PK parameter value exhibiting doseproportionality.

Concentration-dose PK profiles following ingestion of multiples of adose unit can be non-linear. Panel C in FIG. 3 represents an example ofa non-linear, biphasic concentration-dose PK profile. In this example,the biphasic concentration-dose PK profile contains a first phase overwhich the concentration-dose PK profile has a positive rise, and then asecond phase over which the relationship between number of dose unitsingested and a PK parameter value (e.g., drug Cmax) is relatively flat(substantially linear with zero slope). For such a dose unit, forexample, drug Cmax can be increased for a selected number of dose units(e.g., 2, 3, or 4 dose units). However, ingestion of additional doseunits does not provide for a significant increase in drug Cmax.

Panel D in FIG. 3 represents another example of a non-linear, biphasicconcentration-dose PK profile. In this example, the biphasicconcentration-dose PK profile is characterized by a first phase overwhich the concentration-dose PK profile has a positive rise and a secondphase over which the relationship between number of dose units ingestedand a PK parameter value (e.g., drug Cmax) declines. Dose units thatprovide this concentration-dose PK profile provide for an increase indrug Cmax for a selected number of ingested dose units (e.g., 2, 3, or 4dose units). However, ingestion of further additional dose units doesnot provide for a significant increase in drug Cmax and instead providesfor decreased drug Cmax.

Panel E in FIG. 3 represents a concentration-dose PK profile in whichthe relationship between the number of dose units ingested and a PKparameter (e.g., drug Cmax) is linear with zero slope. Such dose unitsdo not provide for a significant increase or decrease in drug Cmax withingestion of multiples of dose units.

Panel F in FIG. 3 represents a concentration-dose PK profile in whichthe relationship between number of dose units ingested and a PKparameter value (e.g., drug Cmax) is linear with a negative slope. Thus,drug Cmax decreases as the number of dose units ingested increases.

Dose units that provide for concentration-dose PK profiles whenmultiples of a dose unit are ingested find use in tailoring of a dosageregimen to provide a therapeutic level of released drug while reducingthe risk of overdose, misuse, or abuse. Such reduction in risk can becompared to a reference, e.g., to administration of drug alone orprodrug alone. In one embodiment, risk is reduced compared toadministration of a drug or prodrug that provides a proportionalconcentration-dose PK profile. A dose unit that provides for aconcentration-dose PK profile can reduce the risk of patient overdosethrough inadvertent ingestion of dose units above a prescribed dosage.Such a dose unit can reduce the risk of patient misuse (e.g., throughself-medication). Such a dose unit can discourage abuse throughdeliberate ingestion of multiple dose units. For example, a dose unitthat provides for a biphasic concentration-dose PK profile can allow foran increase in drug release for a limited number of dose units ingested,after which an increase in drug release with ingestion of more doseunits is not realized. In another example, a dose unit that provides fora concentration-dose PK profile of zero slope can allow for retention ofa similar drug release profile regardless of the number of dose unitsingested.

Ingestion of multiples of a dose unit can provide for adjustment of a PKparameter value relative to that of ingestion of multiples of the samedose (either as drug alone or as a prodrug) in the absence of inhibitorsuch that, for example, ingestion of a selected number (e.g., 2, 3, 4 ormore) of a single dose unit provides for a decrease in a PK parametervalue compared to ingestion of the same number of doses in the absenceof inhibitor.

Pharmaceutical compositions include those having an inhibitor to providefor protection of a therapeutic compound from degradation in the GItract. Inhibitor can be combined with a drug (i.e., not a prodrug) toprovide for protection of the drug from degradation in the GI system. Inthis example, the composition of inhibitor and drug provide for amodified PK profile by increasing a PK parameter. Inhibitor can also becombined with a prodrug that is susceptible to degradation by a GIenzyme and has a site of action outside the GI tract. In thiscomposition, the inhibitor protects ingested prodrug in the GI tractprior to its distribution outside the GI tract and cleavage at a desiredsite of action.

Methods Used to Define Relative Amounts of Prodrug and Inhibitor in aDose Unit

Dose units that provide for a desired PK profile, such as a desiredconcentration-time PK profile and/or a desired concentration-dose PKprofile, can be made by combining a prodrug and an inhibitor in a doseunit in relative amounts effective to provide for release of drug thatprovides for a desired drug PK profile following ingestion by a patient.

Prodrugs can be selected as suitable for use in a dose unit bydetermining the GI enzyme-mediated drug release competency of theprodrug. This can be accomplished in vitro, in vivo or ex vivo.

In vitro assays can be conducted by combining a prodrug with a GI enzyme(e.g., trypsin) in a reaction mixture. The GI enzyme can be provided inthe reaction mixture in an amount sufficient to catalyze cleavage of theprodrug. Assays are conducted under suitable conditions, and optionallymay be under conditions that mimic those found in a GI tract of asubject, e.g., human. “Prodrug conversion” refers to release of drugfrom prodrug. Prodrug conversion can be assessed by detecting a level ofa product of prodrug conversion (e.g., released drug) and/or bydetecting a level of prodrug that is maintained in the presence of theGI enzyme. Prodrug conversion can also be assessed by detecting the rateat which a product of prodrug conversion occurs or the rate at whichprodrug disappears. An increase in released drug, or a decrease inprodrug, indicate prodrug conversion has occurred. Prodrugs that exhibitan acceptable level of prodrug conversion in the presence of the GIenzyme within an acceptable period of time are suitable for use in adose unit in combination with an inhibitor of the GI enzyme that isshown to mediate prodrug conversion.

In vivo assays can assess the suitability of a prodrug for use in a doseunit by administration of the prodrug to an animal (e.g., a human ornon-human animal, e.g., rat, dog, pig, etc.). Such administration can beenteral (e.g., oral administration). Prodrug conversion can be detectedby, for example, detecting a product of prodrug conversion (e.g.,released drug or a metabolite of released drug) or detecting prodrug inblood or plasma of the animal at a desired time point(s) followingadministration.

Ex vivo assays, such as a gut loop or inverted gut loop assay, canassess the suitability of a prodrug for use in a dose unit by, forexample, administration of the prodrug to a ligated section of theintestine of an animal. Prodrug conversion can be detected by, forexample, detecting a product of prodrug conversion (e.g., released drugor a metabolite of released drug) or detecting prodrug in the ligatedgut loop of the animal at a desired time point(s) followingadministration.

Inhibitors are generally selected based on, for example, activity ininteracting with the GI enzyme(s) that mediate release of drug from aprodrug with which the inhibitor is to be co-dosed. Such assays can beconducted in the presence of enzyme either with or without prodrug.Inhibitors can also be selected according to properties such ashalf-life in the GI system, potency, avidity, affinity, molecular sizeand/or enzyme inhibition profile (e.g., steepness of inhibition curve inan enzyme activity assay, inhibition initiation rate). Inhibitors foruse in prodrug-inhibitor combinations can be selected through use of invitro, in vivo and/or ex vivo assays.

One embodiment is a method for identifying a prodrug and a GI enzymeinhibitor suitable for formulation in a dose unit wherein the methodcomprises combining a prodrug (e.g., an amphetamine prodrug), a GIenzyme inhibitor (e.g., a trypsin inhibitor), and a GI enzyme (e.g.,trypsin) in a reaction mixture and detecting prodrug conversion. Such acombination is tested for an interaction between the prodrug, inhibitorand enzyme, i.e., tested to determine how the inhibitor will interactwith the enzyme that mediates enzymatically-controlled release of thedrug from the prodrug. In one embodiment, a decrease in prodrugconversion in the presence of the GI enzyme inhibitor as compared toprodrug conversion in the absence of the GI enzyme inhibitor indicatesthe prodrug and GI enzyme inhibitor are suitable for formulation in adose unit. Such a method can be an in vitro assay.

One embodiment is a method for identifying a prodrug and a GI enzymeinhibitor suitable for formulation in a dose unit wherein the methodcomprises administering to an animal a prodrug (e.g., an amphetamineprodrug) and a GI enzyme inhibitor (e.g., a trypsin inhibitor) anddetecting prodrug conversion. In one embodiment, a decrease in prodrugconversion in the presence of the GI enzyme inhibitor as compared toprodrug conversion in the absence of the GI enzyme inhibitor indicatesthe prodrug and GI enzyme inhibitor are suitable for formulation in adose unit. Such a method can be an in vivo assay; for example, theprodrug and GI enzyme inhibitor can be administered orally. Such amethod can also be an ex vivo assay; for example, the prodrug and GIenzyme inhibitor can be administered orally or to a tissue, such as anintestine, that is at least temporarily exposed. Detection can occur inthe blood or plasma or respective tissue. As used herein, tissue refersto the tissue itself and can also refer to contents within the tissue.

One embodiment is a method for identifying a prodrug and a GI enzymeinhibitor suitable for formulation in a dose unit wherein the methodcomprises administering a prodrug and a gastrointestinal (GI) enzymeinhibitor to an animal tissue that has removed from an animal anddetecting prodrug conversion. In one embodiment, a decrease in prodrugconversion in the presence of the GI enzyme inhibitor as compared toprodrug conversion in the absence of the GI enzyme inhibitor indicatesthe prodrug and GI enzyme inhibitor are suitable for formulation in adose unit.

In vitro assays can be conducted by combining a prodrug, an inhibitorand a GI enzyme in a reaction mixture. The GI enzyme can be provided inthe reaction mixture in an amount sufficient to catalyze cleavage of theprodrug, and assays conducted under suitable conditions, optionallyunder conditions that mimic those found in a GI tract of a subject,e.g., human. Prodrug conversion can be assessed by detecting a level ofa product of prodrug conversion (e.g., released drug) and/or bydetecting a level of prodrug maintained in the presence of the GIenzyme. Prodrug conversion can also be assessed by detecting the rate atwhich a product of prodrug conversion occurs or the rate at whichprodrug disappears. Prodrug conversion that is modified in the presenceof inhibitor as compared to a level of prodrug conversion in the absenceof inhibitor indicates the inhibitor is suitable for attenuation ofprodrug conversion and for use in a dose unit. Reaction mixtures havinga fixed amount of prodrug and increasing amounts of inhibitor, or afixed amount of inhibitor and increasing amounts of prodrug, can be usedto identify relative amounts of prodrug and inhibitor which provide fora desired modification of prodrug conversion.

In vivo assays can assess combinations of prodrugs and inhibitors byco-dosing of prodrug and inhibitor to an animal. Such co-dosing can beenteral. “Co-dosing” refers to administration of prodrug and inhibitoras separate doses or a combined dose (i.e., in the same formulation).Prodrug conversion can be detected by, for example, detecting a productof prodrug conversion (e.g., released drug or drug metabolite) ordetecting prodrug in blood or plasma of the animal at a desired timepoint(s) following administration. Combinations of prodrug and inhibitorcan be identified that provide for a prodrug conversion level thatyields a desired PK profile as compared to, for example, prodrug withoutinhibitor.

Combinations of relative amounts of prodrug and inhibitor that providefor a desired PK profile can be identified by dosing animals with afixed amount of prodrug and increasing amounts of inhibitor, or with afixed amount of inhibitor and increasing amounts of prodrug. One or morePK parameters can then be assessed, e.g., drug Cmax, drug Tmax, and drugexposure. Relative amounts of prodrug and inhibitor that provide for adesired PK profile are identified as amounts of prodrug and inhibitorfor use in a dose unit. The PK profile of the prodrug and inhibitorcombination can be, for example, characterized by a decreased PKparameter value relative to prodrug without inhibitor. A decrease in thePK parameter value of an inhibitor-to-prodrug combination (e.g., adecrease in drug Cmax, a decrease in 1/drug Tmax (i.e., a delay in drugTmax) or a decrease in drug exposure) relative to a corresponding PKparameter value following administration of prodrug without inhibitorcan be indicative of an inhibitor-to-prodrug combination that canprovide a desired PK profile. Assays can be conducted with differentrelative amounts of inhibitor and prodrug.

In vivo assays can be used to identify combinations of prodrug andinhibitor that provide for dose units that provide for a desiredconcentration-dose PK profile following ingestion of multiples of thedose unit (e.g., at least 2, at least 3, at least 4 or more). Ex vivoassays can be conducted by direct administration of prodrug andinhibitor into a tissue and/or its contents of an animal, such as theintestine, including by introduction by injection into the lumen of aligated intestine (e.g., a gut loop, or intestinal loop, assay, or aninverted gut assay). An ex vivo assay can also be conducted by excisinga tissue and/or its contents from an animal and introducing prodrug andinhibitor into such tissues and/or contents.

For example, a dose of prodrug that is desired for a single dose unit isselected (e.g., an amount that provides an efficacious plasma druglevel). A multiple of single dose units for which a relationship betweenthat multiple and a PK parameter to be tested is then selected. Forexample, if a concentration-dose PK profile is to be designed foringestion of 2, 3, 4, 5, 6, 7, 8, 9 or 10 dose units, then the amount ofprodrug equivalent to ingestion of that same number of dose units isdetermined (referred to as the “high dose”). The multiple of dose unitscan be selected based on the number of ingested pills at which drug Cmaxis modified relative to ingestion of the single dose unit. If, forexample, the profile is to provide for abuse deterrence, then a multipleof 10 can be selected, for example. A variety of different inhibitors(e.g., from a panel of inhibitors) can be tested using differentrelative amounts of inhibitor and prodrug. Assays can be used toidentify suitable combination(s) of inhibitor and prodrug to obtain asingle dose unit that is therapeutically effective, wherein such acombination, when ingested as a multiple of dose units, provides amodified PK parameter compared to ingestion of the same multiple of drugor prodrug alone (wherein a single dose of either drug or prodrug alonereleases into blood or plasma the same amount of drug as is released bya single dose unit).

Increasing amounts of inhibitor are then co-dosed to animals with thehigh dose of prodrug. The dose level of inhibitor that provides adesired drug Cmax following ingestion of the high dose of prodrug isidentified and the resultant inhibitor-to-prodrug ratio determined.

Prodrug and inhibitor are then co-dosed in amounts equivalent to theinhibitor-to-prodrug ratio that provided the desired result at the highdose of prodrug. The PK parameter value of interest (e.g., drug Cmax) isthen assessed. If a desired PK parameter value results followingingestion of the single dose unit equivalent, then single dose unitsthat provide for a desired concentration-dose PK profile are identified.For example, where a zero dose linear profile is desired, the drug Cmaxfollowing ingestion of a single dose unit does not increasesignificantly following ingestion of a multiple number of the singledose units.

Methods for Manufacturing, Formulating, and Packaging Dose Units

Dose units of the present disclosure can be made using manufacturingmethods available in the art and can be of a variety of forms suitablefor enteral (including oral, buccal and sublingual) administration, forexample as a tablet, capsule, thin film, powder, suspension, solution,syrup, dispersion or emulsion. The dose unit can contain componentsconventional in pharmaceutical preparations, e.g. one or more carriers,binders, lubricants, excipients (e.g., to impart controlled releasecharacteristics), pH modifiers, flavoring agents (e.g., sweeteners),bulking agents, coloring agents or further active agents. Dose units ofthe present disclosure can include can include an enteric coating orother component(s) to facilitate protection from stomach acid, wheredesired.

Dose units can be of any suitable size or shape. The dose unit can be ofany shape suitable for enteral administration, e.g., ellipsoid,lenticular, circular, rectangular, cylindrical, and the like.

Dose units provided as dry dose units can have a total weight of fromabout 1 microgram to about 1 gram, and can be from about 5 micrograms to1.5 grams, from about 50 micrograms to 1 gram, from about 100 microgramsto 1 gram, from 50 micrograms to 750 milligrams, and may be from about 1microgram to 2 grams.

Dose units can comprise components in any relative amounts. For example,dose units can be from about 0.1% to 99% by weight of active ingredients(i.e., prodrug and inhibitor) per total weight of dose unit (0.1% to 99%total combined weight of prodrug and inhibitor per total weight ofsingle dose unit). In some embodiments, dose units can be from 10% to50%, from 20% to 40%, or about 30% by weight of active ingredients pertotal weight dose unit.

Dose units can be provided in a variety of different forms andoptionally provided in a manner suitable for storage. For example, doseunits can be disposed within a container suitable for containing apharmaceutical composition. The container can be, for example, a bottle(e.g., with a closure device, such as a cap), a blister pack (e.g.,which can provide for enclosure of one or more dose units per blister),a vial, flexible packaging (e.g., sealed Mylar or plastic bags), anampule (for single dose units in solution), a dropper, thin film, a tubeand the like.

Containers can include a cap (e.g., screw cap) that is removablyconnected to the container over an opening through which the dose unitsdisposed within the container can be accessed.

Containers can include a seal which can serve as a tamper-evident and/ortamper-resistant element, which seal is disrupted upon access to a doseunit disposed within the container. Such seal elements can be, forexample, a frangible element that is broken or otherwise modified uponaccess to a dose unit disposed within the container. Examples of suchfrangible seal elements include a seal positioned over a containeropening such that access to a dose unit within the container requiresdisruption of the seal (e.g., by peeling and/or piercing the seal).Examples of frangible seal elements include a frangible ring disposedaround a container opening and in connection with a cap such that thering is broken upon opening of the cap to access the dose units in thecontainer.

Dry and liquid dose units can be placed in a container (e.g., bottle orpackage, e.g., a flexible bag) of a size and configuration adapted tomaintain stability of dose units over a period during which the doseunits are dispensed into a prescription. For example, containers can besized and configured to contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100or more single dry or liquid dose units. The containers can be sealed orresealable. The containers can packaged in a carton (e.g., for shipmentfrom a manufacturer to a pharmacy or other dispensary). Such cartons canbe boxes, tubes, or of other configuration, and may be made of anymaterial (e.g., cardboard, plastic, and the like). The packaging systemand/or containers disposed therein can have one or more affixed labels(e.g., to provide information such as lot number, dose unit type,manufacturer, and the like).

The container can include a moisture barrier and/or light barrier, e.g.,to facilitate maintenance of stability of the active ingredients in thedose units contained therein. Where the dose unit is a dry dose unit,the container can include a desiccant pack which is disposed within thecontainer. The container can be adapted to contain a single dose unit ormultiples of a dose unit. The container can include a dispensing controlmechanism, such as a lock out mechanism that facilitates maintenance ofdosing regimen.

The dose units can be provided in solid or semi-solid form, and can be adry dose unit. “Dry dose unit” refers to a dose unit that is in otherthan in a completely liquid form. Examples of dry dose units include,for example, tablets, capsules (e.g., solid capsules, capsulescontaining liquid), thin film, microparticles, granules, powder and thelike. Dose units can be provided as liquid dose units, where the doseunits can be provided as single or multiple doses of a formulationcontaining prodrug and inhibitor in liquid form. Single doses of a dryor liquid dose unit can be disposed within a sealed container, andsealed containers optionally provided in a packaging system, e.g., toprovide for a prescribed number of doses, to provide for shipment ofdose units, and the like.

Dose units can be formulated such that the prodrug and inhibitor arepresent in the same carrier, e.g., solubilized or suspended within thesame matrix. Alternatively, dose units can be composed of two or moreportions, where the prodrug and inhibitor can be provided in the same ordifferent portions, and can be provided in adjacent or non-adjacentportions.

Dose units can be provided in a container in which they are disposed,and may be provided as part of a packaging system (optionally withinstructions for use). For example, dose units containing differentamounts of prodrug can be provided in separate containers, whichcontainers can be disposed with in a larger container (e.g., tofacilitate protection of dose units for shipment). For example, one ormore dose units as described herein can be provided in separatecontainers, where dose units of different composition are provided inseparate containers, and the separate containers disposed within packagefor dispensing.

In another example, dose units can be provided in a double-chambereddispenser where a first chamber contains a prodrug formulation and asecond chamber contains an inhibitor formulation. The dispenser can beadapted to provide for mixing of a prodrug formulation and an inhibitorformulation prior to ingestion. For example, the two chambers of thedispenser can be separated by a removable wall (e.g., frangible wall)that is broken or removed prior to administration to allow mixing of theformulations of the two chambers. The first and second chambers canterminate into a dispensing outlet, optionally through a common chamber.The formulations can be provided in dry or liquid form, or a combinationthereof. For example, the formulation in the first chamber can be liquidand the formulation in the second chamber can be dry, both can be dry,or both can be liquid.

Dose units that provide for controlled release of prodrug, of inhibitor,or of both prodrug and inhibitor are contemplated by the presentdisclosure, where “controlled release” refers to release of one or bothof prodrug and inhibitor from the dose unit over a selected period oftime and/or in a pre-selected manner.

Methods of Use of Dose Units

Dose units are advantageous because they find use in methods to reduceside effects and/or improve tolerability of drugs to patients in needthereof by, for example, limiting a PK parameter as disclosed herein.The present disclosure thus provides methods to reduce side effects byadministering a dose unit of the present disclosure to a patient in needso as to provide for a reduction of side effects as compared to thoseassociated with administration of drug and/or as compared toadministration of prodrug without inhibitor. The present disclosure alsoprovides methods to improve tolerability of drugs by administering adose unit of the present disclosure to a patient in need so as toprovide for improvement in tolerability as compared to administration ofdrug and/or as compared to administration of prodrug without inhibitor.

Dose units find use in methods for increasing patient compliance of apatient with a therapy prescribed by a clinician, where such methodsinvolve directing administration of a dose unit described herein to apatient in need of therapy so as to provide for increased patientcompliance as compared to a therapy involving administration of drugand/or as compared to administrations of prodrug without inhibitor. Suchmethods can help increase the likelihood that a clinician-specifiedtherapy occurs as prescribed.

Dose units can provide for enhanced patient compliance and cliniciancontrol. For example, by limiting a PK parameter (e.g., such as drugCmax or drug exposure) when multiples (e.g., two or more, three or more,or four or more) dose units are ingested, a patient requiring a higherdose of drug must seek the assistance of a clinician. The dose units canprovide for control of the degree to which a patient can readily“self-medicate”, and further can provide for the patient to adjust doseto a dose within a permissible range. Dose units can provide for reducedside effects, by for example, providing for delivery of drug at anefficacious dose but with a modified PK profile over a period oftreatment, e.g., as defined by a decreased PK parameter (e.g., decreaseddrug Cmax, decreased drug exposure).

Dose units find use in methods to reduce the risk of unintended overdoseof drug that can follow ingestion of multiple doses taken at the sametime or over a short period of time. Such methods of the presentdisclosure can provide for reduction of risk of unintended overdose ascompared to risk of unintended overdose of drug and/or as compared torisk of unintended overdose of prodrug without inhibitor. Such methodsinvolve directing administration of a dosage described herein to apatient in need of drug released by conversion of the prodrug. Suchmethods can help avoid unintended overdosing due to intentional orunintentional misuse of the dose unit.

The present disclosure provides methods to reduce misuse and abuse of adrug, as well as to reduce risk of overdose, that can accompanyingestion of multiples of doses of a drug, e.g., ingested at the sametime. Such methods generally involve combining in a dose unit a prodrugand an inhibitor of a GI enzyme that mediates release of drug from theprodrug, where the inhibitor is present in the dose unit in an amounteffective to attenuate release of drug from the prodrug, e.g., followingingestion of multiples of dose units by a patient. Such methods providefor a modified concentration-dose PK profile while providingtherapeutically effective levels from a single dose unit, as directed bythe prescribing clinician. Such methods can provide for, for example,reduction of risks that can accompany misuse and/or abuse of a prodrug,particularly where conversion of the prodrug provides for release of anarcotic or other drug of abuse (e.g., amphetamine). For example, whenthe prodrug provides for release of a drug of abuse, dose units canprovide for reduction of reward that can follow ingestion of multiplesof dose units of a drug of abuse.

Dose units can provide clinicians with enhanced flexibility inprescribing drug. For example, a clinician can prescribe a dosageregimen involving different dose strengths, which can involve two ormore different dose units of prodrug and inhibitor having differentrelative amounts of prodrug, different amounts of inhibitor, ordifferent amounts of both prodrug and inhibitor. Such different strengthdose units can provide for delivery of drug according to different PKparameters (e.g., drug exposure, drug Cmax, and the like as describedherein). For example, a first dose unit can provide for delivery of afirst dose of drug following ingestion, and a second dose unit canprovide for delivery of a second dose of drug following ingestion. Thefirst and second prodrug doses of the dose units can be differentstrengths, e.g., the second dose can be greater than the first dose. Aclinician can thus prescribe a collection of two or more, or three ormore dose units of different strengths, which can be accompanied byinstructions to facilitate a degree of self-medication, e.g., toincrease delivery of an amphetamine drug according to a patient's needsto treat ADHD, CFS, brain injury, narcolepsy, or obesity.

Thwarting Tampering by Trypsin Mediated Release of Amphetamine fromProdrugs

The disclosure provides for a composition comprising a compounddisclosed herein and a trypsin inhibitor that reduces drug abusepotential. A trypsin inhibitor can thwart the ability of a user to applytrypsin to effect the release of amphetamine from the amphetamineprodrug in vitro. For example, if an abuser attempts to incubate trypsinwith a composition of the embodiments that includes an amphetamineprodrug and a trypsin inhibitor, the trypsin inhibitor can reduce theaction of the added trypsin, thereby thwarting attempts to releaseamphetamine for purposes of abuse.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the embodiments, and are not intended to limit the scope ofwhat the inventors regard as their invention nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Celsius, and pressure is ator near atmospheric. Standard abbreviations may be used.

Example 1 Synthesis of Amphetamine Prodrug Compounds Synthesis ofAmphetamine-arginine-acetate (Compound AM-1; also referred to as2-acetylamino-5-guanidino-pentanoic acid((R)-1-methyl-2-phenyl-ethyl)-amide) and Amphetamine-arginine-malonicacid (Compound AM-2; also referred to asN-[4-guanidino-1-((R)-1-methyl-2-phenyl-ethylcarbamoyl)-butyl]-malonicacid)

Preparation of Compound A

D-Amphetamine sulfate (5.0 g, 27.1 mmol), Boc-Arg(Pbf)-OH (10.0 g, 19.0mmol) and HATU (10.8 g, 28.5 mmol) were suspended in DMF (100 mL),brought to 5° C. and treated drop wise with DIEA (13.3 mL, 76.0 mmol)over 10 min. The reaction mixture was stirred at 5° C. for an additional10 min, warmed to ambient temperature, followed by stirring for 30 min.The reaction was then diluted with EtOAc (400 mL) and poured into water(600 mL). The layers were separated, the aqueous layer extracted withEtOAc (3×300 mL) and the combined organic layers washed with 2% aq.H₂SO₄ (150 mL), water (2×600 mL) and brine (600 mL). The organic layerwas dried over MgSO₄, filtered and concentrated in vacuo to givecompound A in quantitative yield (13.9 g, 19.0 mmol) as a light-yellowfoamy solid. LC-MS [M+H] 644.7 (C₃₃H₄₉N₅O₆S+H, calc: 644.8). Compound Awas used without further purification.

Preparation of Compound B

A solution of compound A (13.9 g, 19.0 mmol) in DCM (80 mL) was treatedwith 4 M HCl in dioxane (48 mL, 190 mmol) and the mixture stirred atambient temperature for 45 min. Ether (1 L) was added and the resultingwhite precipitate filtered, washed with ether (50 mL), hexane (50 mL)and then dried in vacuo to give compound B as an off-white solid inquantitative yield (11.9 g, 19.0 mmol). LC-MS [M+H] 544.4(C₂₈H₄₁N₅O₄S+H, calc: 544.7). Compound B was used without furtherpurification.

Preparation of Compound C

To a cooled solution (5° C.) of compound B (11.9 g, 19.0 mmol) and monotert-butyl malonate (3.2 g, 20.0 mmol) in DMF (70 mL) was added portionwise, BOP (9.0 g, 20.3 mmol) over 5 min. This step was then followed bydrop wise addition of DIEA (13.3 mL, 76.0 mmol) over 15 min. Stirringwas continued for an additional 15 min, after which the ice bath wasremoved and the mixture warmed to ambient temperature. After 30 min atambient temperature, the reaction mixture was diluted with EtOAc (600mL) and poured into water (600 mL). The layers were separated and theaqueous layer extracted with EtOAc (2×300 mL). The combined organiclayers were washed with 2% aq. H₂SO₄ (150 mL), water (2×450 mL) andbrine (400 mL), followed by drying over MgSO₄, filtration, and finallythe solvent was evaporated in vacuo. The resulting residue was dried invacuo to give compound C in 93% yield (12.1 g, 17.6 mmol) as a yellowishfoamy solid. LC-MS [M+H] 686.5 (C₃₅H₅₁N₅O₇S+H, calc: 685.9). Compound Cwas used without further purification.

Preparation of Compound AM-2

A solution of compound C (12.1 g, 17.6 mmol) in 5% m-cresol/TFA (350 mL)was stirred at ambient temperature. After 45 min, the solvent wasremoved in vacuo until about 100 mL volume remained, followed bydilution with hexane (500 mL). The solvent was decanted off, and theoily precipitate was concentrated in vacuo. The residue was dissolved in0.1% TFA/H₂O (125 mL), sonicated for 30 min, and the layers separated.The aqueous emulsion (140 mL total volume) cleared up after standing inthe refrigerator overnight. This product was divided into four portions,and each was subjected to HPLC purification [Nanosyn-Pack Microsorb(100-10) C-18 column (50×300 mm); flow rate: 100 mL/min; injectionvolume: 40 mL; mobile phase A: 100% water, 0.1% TFA; mobile phase B:100% ACN, 0.1% TFA; isocratic elution at 0% B in 2 min, gradient elutionto 8% B in 12 min, isocratic elution at 8% B in 30 min, gradient elutionfrom 8% B to 33% B in 51 min; detection at UV 254 nm]. Fractionscontaining the desired compound were combined and concentrated in vacuoto yield the TFA salt of Compound AM-2 in 38% yield (4.0 g, 6.7 mmol,96% purity) as colorless viscous oil. A part of this material (640 mg,1.06 mmol) was dissolved in i-PrOH (3 mL) and treated with 2 N HCl inether (30 mL, 60 mmol) to give the hydrochloride salt of Compound AM-2(430 mg, 0.95 mmol, 99% purity). LC-MS [M+H] 378.3 (C₁₈H₂₇N₅O₄+H, calc:378.4). Retention time [Chromolith SpeedRod RP-18e C18 column (4.6×50mm); flow rate: 1.5 ml/min; mobile phase A: 0.1% TFA/water; mobile phaseB 0.1% TFA/ACN; gradient elution from 5% B to 100% B over 9.6 min,detection 254 nm]: 2.15 min.

Preparation of Compound D

To a solution of compound B (3.3 g, 5.0 mmol) in chloroform (30 mL) wasadded DIEA (2.3 mL, 13.1 mmol) and acetic anhydride (697 mg, 6.8 mmol).After stirring at ambient temperature for 30 min, the mixture wastreated with 2 M EtNH₂ in THF (2.8 mL, 1.8 mmol). Stirring was continuedfor an additional 30 min. The solvent was then removed in vacuo, and theresidue acidified to pH ˜3 with 2% aq. H₂SO₄ and extracted with EtOAc(3×100 mL). The combined organic layers were washed with water (150 mL),sat. NaHCO₃ solution (150 mL) and brine (150 mL). After drying overMgSO₄, the solvent was evaporated in vacuo to give compound D in 95%yield (2.8 g, 4.8 mmol) as a colorless foamy solid. LC-MS [M+H] 586.2(C₃₀H₄₃N₅O₅S+H, calc: 586.8). Compound D was used without furtherpurification.

Preparation of Compound AM-1

A solution of compound D (2.8 g, 4.8 mmol) in 5% m-cresol/TFA (70 mL)was stirred at ambient temperature. After 45 min, TFA was evaporated andthe residue taken into MeOH (10 mL), diluted with hexane (400 mL) andcooled in the refrigerator (4° C.) for 30 min. After separation from thehexane layer, the oily precipitate was dissolved in water (30 mL) andpurified by HPLC. [Nanosyn-Pack Microsorb (100-10) C-18 column (50×300mm); flow rate: 100 mL/min; injection volume: 35 mL; mobile phase A:100% water, 0.1% TFA; mobile phase B: 100% ACN, 0.1% TFA; isocraticelution at 5% B in 5 min, gradient elution to 12% B in 7 min, isocraticelution at 12% B in 20 min, gradient elution from 12% B to 40% B in 28min; detection at UV 254 nm]. Fractions containing the desired compoundwere combined and concentrated in vacuo. Traces of water were removed bydissolving the residue in i-PrOH (50 mL) followed by evaporation invacuo (procedure was repeated twice). The residue was dissolved ini-PrOH (20 mL) and treated with 2 N HCl in ether (100 mL, 200 mmol) togive the hydrochloride salt of Compound AM-1 in 76% yield (1.34 g, 3.6mmol, 99% purity) LC-MS [M+H] 334.4 (C₁₇H₂₇N₅O₂+H, calc: 334.4).Retention time [Chromolith SpeedRod RP-18e C18 column (4.6×50 mm); flowrate: 1.5 ml/min; mobile phase A: 0.1% TFA/water; mobile phase B 0.1%TFA/ACN; gradient elution from 5% B to 100% B over 9.6 min, detection254 nm]: 2.43 min.

Example 2: Synthesis of Amphetamine-arginine-N-methyl (Compound AM-4)

Preparation of Compound E

D-Amphetamine sulfate (496 mg, 2.68 mmol), Boc-N-Me-Arg(Mtr)-OH (940 mg,1.88 mmol) and HATU (855 mg, 2.25 mmol) were suspended in DMF (20 mL),cooled to 5° C. and treated drop wise with DIEA (1.9 mL, 10.7 mmol) over5 min. After addition, the reaction mixture was stirred at 5° C. for anadditional 10 min. Next, the reaction was warmed to ambient temperatureand stirred for 30 min. The reaction was then diluted with EtOAc (250mL) and poured into water (200 mL). The layers were separated, theaqueous layer extracted with EtOAc (2×150 mL), and the combined organiclayers washed with 2% aq. H₂SO₄ (50 mL), water (2×300 mL) and brine (200mL). The organic layer was dried over MgSO₄, filtered and concentratedin vacuo to give compound E in quantitative yield (1.28 g, 1.88 mmol) asa colorless foamy solid. LC-MS [M+H] 618.6 (C₃₁H₄₇N₅O₆S+H, calc: 618.3).Compound E was used without further purification.

Preparation of Compound AM-4

A solution of compound E (1.28 g, 1.88 mmol) in 5% m-cresol/TFA (50 mL)was stirred at ambient temperature. After 6 h, the solvent was removedin vacuo until about 15 mL remained. The mixture was diluted with hexane(300 mL) and resulted in the formation of an oily precipitate. Hexanewas decanted off and the oily precipitate was concentrated in vacuo toremove traces of hexane. The residue was dissolved in 0.1% TFA/H₂O (25mL), sonicated for 10 min and subjected to HPLC purification[Nanosyn-Pack Microsorb (100-10) C-18 column (50×300 mm); flow rate: 100mL/min; injection volume: 28 mL; mobile phase A: 100% water, 0.1% TFA;mobile phase B: 100% ACN, 0.1% TFA; gradient elution from 0 to 10% B in10 min, isocratic elution at 10% B in 25 min, gradient elution from 10%B to 42% B in 55 min; detection at UV 254 nm]. Fractions containing thedesired compound were combined and concentrated in vacuo. The residuewas dissolved in toluene (30 mL, to remove traces of water) andco-evaporated in vacuo (procedure repeated twice). The residue wasdissolved in ACN (5 mL), treated with 2.0 M HCl in ether (50 mL)followed by dilution with ether (200 mL). The resulting solid wasfiltered, washed with ether (3×30 mL) and hexane (40 mL) and dried invacuo to provide the hydrochloride salt of Compound AM-4 in 48% yield(400.6 mg, 0.90 mmol) as a white solid. LC-MS [M+H] 306.4 (C₁₆H₂₇N₅O+H,calc: 306.2). Purity ˜94% (UV/254 nm).

Example 3: Synthesis of Amphetamine-lysine-acetate (Compound AM-5)

Compound AM-5 was prepared following the method described in Example 1to prepare Amphetamine-arginine-acetate (Compound AM-1), but usingFmoc-Lys(Boc)-OH instead of Boc-Arg(Pbf)-OH, piperidine instead of HClin dioxane (for deprotection of Fmoc group on ca-nitrogen of Lys), andlastly HCl in dioxane for Boc deprotection of the Lys reside. LC-MS[M+H] 306.6 (C₁₇H₂₈N₃O₂+H, calc: 306.2).

Example 4: Synthesis of Amphetamine-Arginine (Compound AM-6)

Compound AM-6 was prepared following the method described in Example 1to prepare Amphetamine-arginine-acetate (Compound AM-1), but skippingthe step involving the use of acetic anhydride. LC-MS [M+H] 291.4(C₁₅H₂₆N₅O+H, calc: 292.2).

Biological Data

Example 5: In Vitro Trypsin Conversion of Prodrugs to Amphetamine andInhibition by Trypsin Inhibitor

This Example demonstrates the ability of trypsin to cleave a prodrug ofthe embodiments to effect amphetamine release and the effect of trypsininhibitors on such cleavage. This Example also determines the ability oftrypsin to cleave lisdexamphetamine.

Compound AM-1 and Compound AM-2 (each of which can be prepared asdescribed in Examples herein) and Compound AM-3 (lisdexamphetamine,available from Shire Pharmaceuticals, Wayne, Pa. (Vyvanse®)) were eachincubated with trypsin from bovine pancreas (Catalog No. T8003, Type I,˜10,000 BAEE units/mg protein, Sigma-Aldrich, St. Louis, Mo.) in theabsence or presence of trypsin inhibitor, Compound 109 (Catalog No.3081, Tocris Bioscience, Ellisville, Mo.). The reactions included 0.761mM Compound AM-1.HCl, Compound AM-2.HCl or Compound AM-3.HCl in thepresence of 0.0228 mg/ml trypsin, 22.5 mM calcium chloride, 172 mM TrispH 8 and either 0.25% DMSO or Compound 109 as indicated in Table 1,depending on whether inhibitor was included in the incubation. Thereactions were conducted at 37° C. for 24 hr. When Compound 109 was partof the incubation mixture, the respective prodrug was added 5 min afterthe other incubation components. Samples were collected at specifiedtime points, transferred into 0.5% formic acid in acetonitrile to stoptrypsin activity and stored at less than −70° C. until analysis byLC-MS/MS.

Table 1 indicates the results of exposure of the test compounds totrypsin in the absence or presence of trypsin inhibitor. The results areexpressed as half-life of prodrug when exposed to trypsin (i.e., prodrugtrypsin half-life) in hours and rate of amphetamine formulation inumols/h/U BAEE.

TABLE 1 In vitro trypsin conversion of prodrugs to amphetamine andinhibition by trypsin inhibitor No trypsin inhibitor With trypsininhibitor Pro-drug Rate of AMP Pro-drug Rate of AMP trypsin formation,trypsin formation, half-life, h umols/h/UBAEE Compound half-life, humols/h/UBAEE Prodrug Ave ± sd Ave ± sd 109 Ave ± sd Ave ± sd AM-1 39.9± 0.5 0.0455 ± 0.0102 2 uM 54.0 ± 4.6  nd AM-2  59.9 ± 17.1 0.0310 ±0.0004 2 uM 68.5 ± 16.4 nd AM-3 112 ± 14 nd 2 uM 393 ± 431 nd nd = notdetectable

The results in Table 1 indicated that trypsin released amphetamine fromCompound AM-1 and Compound AM-2 and that a trypsin inhibitor of theembodiments attenuated trypsin-mediated release of amphetamine. The rateof formation of amphetamine from Compound AM-3 was not detectableshowing that trypsin did not efficiently release amphetamine fromCompound AM-3.

Example 6: Pharmacokinetics of Compound AM-1 Following PO Administrationto Rats

This Example compares the pharmacokinetics of two concentrations ofCompound AM-1 administered orally (PO) to rats.

Saline solutions of Compound AM-1 (which can be prepared as described inExamples herein) were dosed as indicated in Table 2 via oral gavage intojugular vein-cannulated male Sprague Dawley rats (4 per group) that hadbeen fasted for 16-18 hr prior to oral dosing. At specified time points,blood samples were drawn, harvested for plasma via centrifugation at5,400 rpm at 4° C. for 5 min, and 100 microliters (l) plasma transferredfrom each sample into a fresh tube containing 2 μl of 50% formic acid.The tubes were vortexed for 5-10 seconds, immediately placed in dry iceand then stored in a −80° C. freezer until analysis by HPLC/MS.

Table 2, FIG. 4(a) and FIG. 4(b) provide amphetamine exposure resultsfor rats administered with different doses of Compound AM-1. Table 2 andFIG. 4(b) also provide prodrug exposure results for rats administeredthe higher dose of Compound AM-1. Results in Table 2 are reported, foreach group of 4 rats, as (a) maximum plasma concentration (Cmax) ofprodrug Compound AM-1 (average±standard deviation), (b) time afteradministration of Compound AM-1 to reach maximum prodrug Compound AM-1concentration (Tmax) (average±standard deviation), (c) maximum plasmaconcentration (Cmax) of amphetamine (AMP) (average±standard deviation)and (d) time after administration of Compound AM-1 to reach maximumamphetamine concentration (Tmax) (average±standard deviation).

TABLE 2 Cmax and Tmax values of prodrug Compound AM-1 and amphetamine inrat plasma Prodrug Prodrug AM-1 AM-1 AMP Dose, Dose Cmax ± Tmax ± Cmax ±AMP Com- mg/ μmol/ sd, sd, sd, Tmax ± pound kg kg ng/mL hr ng/mL sd, hrAM-1  6 16 na na 64.2 ± 10 1.0 ± 0.0 AM-1 24 65 1.28 ± 0.81 0.625 ± 0.43 248 ± 40 2.0 ± 0.8Lower limit of quantitation for AMP for the 6-mg/kg dose was 1.000ng/mL.Lower limits of quantitation for prodrug and AMP for the 24-mg/kg dosewere 0.100 and 0.500 ng/ml, respectively.na=not analyzed

FIG. 4(a) compares mean plasma concentrations over time of amphetaminerelease following PO administration of increasing doses of CompoundAM-1. FIG. 4(b) compares mean plasma concentrations over time of prodrugdisappearance and of amphetamine release following PO administration ofCompound AM-1.

The results in Table 2, FIG. 4(a) and FIG. 4(b) indicate that plasmaconcentrations of amphetamine increased proportionally with CompoundAM-1 dose. The results further indicated that the mean concentration ofprodrug in plasma following oral administration of Compound AM-1 wasvery low, particularly as compared to the concentrations of CompoundAM-2 (see Example 10) or lisdexamphetamine (see Example 14).

Example 7: Oral Administration of Compound AM-1 and Trypsin InhibitorCompound 109 to Rats

This Example demonstrates the ability of a trypsin inhibitor of theembodiments to affect drug release into plasma from Compound AM-1administered orally.

Saline solutions of Compound AM-1 (which can be prepared as described inExamples herein) were dosed at 16 μmol/kg (6 mg/kg) with or without aco-dose of 55 μmol/kg (30 mg/kg) Compound 109 (Catalog No. 3081, TocrisBioscience or Catalog No. WS38665, Waterstone Technology, Carmel, Ind.)as indicated in Table 3 via oral gavage into jugular vein-cannulatedmale Sprague Dawley rats (4 per group) that had been fasted for 16-18 hrprior to oral dosing. At specified time points, blood samples weredrawn, harvested for plasma via centrifugation at 5,400 rpm at 4° C. for5 min, and 100 μl plasma transferred from each sample into a fresh tubecontaining 2 μl of 50% formic acid. The tubes were vortexed for 5-10seconds, immediately placed in dry ice and then stored in a −80° C.freezer until analysis by HPLC/MS.

Table 3 and FIG. 5 provide amphetamine exposure results for ratsadministered with Compound AM-1 and with or without a co-dose of trypsininhibitor. Results in Table 3 are reported, for each group of 4 rats, as(a) maximum plasma concentration (Cmax) of amphetamine (AMP)(average±standard deviation) and (b) time after administration ofCompound AM-1, to reach maximum amphetamine concentration (Tmax)(average±standard deviation).

TABLE 3 Cmax and Tmax values of amphetamine in rat plasma AM-1 AM-1Compound Compound AMP Dose, Dose, 109 Dose, 109 Dose, Cmax ± Tmax ±mg/kg μmol/kg mg/kg μmol/kg sd, ng/mL sd, hr 6 16 0 0 64.2 ± 10  1.0 ±0.0 6 16 30 55 2.08 ± 2.4 6.5 ± 2.1 Lower limit of quantitation was1.000 ng/mL.

FIG. 5 compares mean plasma concentrations over time of amphetaminerelease following PO administration of Compound AM-1 with or without aco-dose of trypsin inhibitor.

The results in Table 3 and FIG. 5 indicated Compound 109's ability toattenuate Compound AM-1's ability to release amphetamine both bysuppressing Cmax and by delaying Tmax.

Example 8: Pharmacokinetics of Compound AM-1 Following IV Administrationto Rats

This Example compares the plasma concentrations of prodrug andamphetamine in rats following intravenous (IV) administration ofCompound AM-1.

Compound AM-1 (which can be prepared as described in Examples herein)was dissolved in saline and injected into the tail vein of 4 jugularvein-cannulated male Sprague Dawley rats at a dose of 5 mg/kg. Atspecified time points, blood samples were drawn, harvested for plasmavia centrifugation at 5,400 rpm at 4° C. for 5 min, and 100 μl plasmatransferred from each sample into a fresh tube containing 2 μl of 50%formic acid. The tubes were vortexed for 5-10 seconds, immediatelyplaced in dry ice and then stored in a −80° C. freezer until analysis byhigh performance liquid chromatography/mass spectrometry (HPLC/MS).

Table 4 and FIG. 6 provide Compound AM-1 and amphetamine exposureresults for the group of rats administered Compound AM-1 intravenously.Results in Table 4 are reported as maximum plasma concentration (Cmax)values (average±standard deviation) of Compound AM-1 and amphetamine,respectively.

TABLE 4 Cmax values of Compound AM-1 and amphetamine in rat plasma AM-1AM-1 AM-1 AMP Dose, Dose, Cmax ± Cmax ± Compound mg/kg μmol/kg sd,ng/mL* sd, ng/mL{circumflex over ( )} AM-1 5 13.5 3250 ± 310 7.48 ± 1.7*Lower limit of quantitation was 0.100 ng/mL {circumflex over ( )}Lowerlimit of quantitation was 0.500 ng/mL

FIG. 6 compares mean plasma concentrations over time of Compound AM-1and amphetamine following IV administration of Compound AM-1 to rats.

Table 4 and FIG. 6 demonstrate that the plasma concentration ofamphetamine in rats administered Compound AM-1 intravenously is only0.23% of the plasma concentration of Compound AM-1, indicating that IVadministration of Compound AM-1 does not lead to significant release ofamphetamine.

Example 9: In Vivo Tolerability of Compound AM-1 in Rats

This Example demonstrates that Compound AM-1 was tolerated whenadministered intravenously to rats.

Male naïve Sprague-Dawley rats, 4 per dose, were used in the study. Ratswere weighed, and then placed under a heat lamp for 15-20 minutes todilate the lateral tail veins. Dose volumes are based on the bodyweights (1 mL/kg); dosing of Compound AM-1 (which can be prepared asdescribed in Example 1 above) was as indicated in Table 5. Beforedosing, rats were placed in Broome restrainers and the drug wasintroduced into one of the tail veins using a syringe and needle. Afterdosing, the timer was set and rats were observed for clinical signs.Blood samples were collected 5 minutes post-dose via the saphenous vein.The rats were observed up to 24 hours. Results are shown in Table 5.

TABLE 5 In vivo tolerability of Compound AM-1 in rats Dose, Dose, Numberof Compound mg/kg μmol/kg Rats dosed Clinical observations AM-1 32 87 4Whole body tremors for 13-30 sec; followed by hypoactivity, then normalafter 2 min

The results in Table 5 indicated that rats tolerate a dose of 87 μmol/kgof Compound AM-1 and recover to normal activity within 2 minutes.

Example 10: Pharmacokinetics of Compound AM-2 Following POAdministration to Rats

This Example compares the pharmacokinetics of two concentrations ofCompound AM-2 administered orally (PO) to rats.

Saline solutions of Compound AM-2 (which can be prepared as described inExample 1 above) were dosed as indicated in Table 6 via oral gavage intojugular vein-cannulated male Sprague Dawley rats (4 per group) that hadbeen fasted for 16-18 hr prior to oral dosing. At specified time points,blood samples were drawn, harvested for plasma via centrifugation at5,400 rpm at 4° C. for 5 min, and 100 μl plasma transferred from eachsample into a fresh tube containing 2 μl of 50% formic acid. The tubeswere vortexed for 5-10 seconds, immediately placed in dry ice and thenstored in −80° C. freezer until analysis by HPLC/MS.

Table 6, FIG. 7(a) and FIG. 7(b) provide amphetamine exposure resultsfor rats administered with different doses of Compound AM-2. Table 6 andFIG. 7(b) also provide prodrug exposure results for rats administeredthe higher dose of Compound AM-2. Results in Table 6 are reported, foreach group of 4 rats, as (a) maximum plasma concentration (Cmax) ofprodrug Compound AM-2 (average±standard deviation), (b) time afteradministration of Compound AM-2 to reach maximum prodrug Compound AM-2concentration (Tmax) (average±standard deviation), (c) maximum plasmaconcentration (Cmax) of amphetamine (AMP) (average±standard deviation)and (d) time after administration of Compound AM-2 to reach maximumamphetamine concentration (Tmax) (average±standard deviation).

TABLE 6 Cmax and Tmax values of prodrug Compound AM-2 and amphetamine inrat plasma Prodrug Prodrug AM-2 AM-2 AMP Dose, Dose Cmax ± Tmax ± Cmax ±AMP Com- mg/ μmol/ sd, sd, sd, Tmax ± pound kg kg ng/mL hr ng/mL sd, hrAM-2 6 14.5 na na 50.6 ± 6.3 2.0 ± 0.0 AM-2 27 65 811 ± 850 0.625 ± 0.25127 ± 31 3.0 ± 0.0 Lower limit of quantitation for AMP for the 6-mg/kgdose was 1.000 ng/mL. Lower limits of quantitation for prodrug and AMPfor the 27-mg/kg dose were 0.100 and 0.500 ng/ml, respectively. na = notanalyzed

FIG. 7(a) compares mean plasma concentrations over time of amphetaminerelease following PO administration of increasing doses of CompoundAM-2. FIG. 7(b) compares mean plasma concentrations over time of prodrugdisappearance and of amphetamine release following PO administration ofCompound AM-2.

The results in Table 6, FIG. 7(a) and FIG. 7(b) indicate that plasmaconcentrations of amphetamine increase proportionally with Compound AM-2dose. The results further indicate that, when both compounds wereadministered orally at 65 μmol/kg, the mean concentration of prodrugCompound AM-2 in plasma was significantly higher than that of CompoundAM-1 (see Example 6).

Example 11: Oral Administration of Compound AM-2 and Trypsin InhibitorCompound 109 to Rats

This Example demonstrates the ability of a trypsin inhibitor of theembodiments to affect drug release into plasma from Compound AM-2administered orally.

Saline solutions of Compound AM-2 (which can be prepared as described inExamples herein) were dosed at 16 μmol/kg (6 mg/kg) with or without aco-dose of 55 μmol/kg (30 mg/kg) Compound 109 (Catalog No. 3081, TocrisBioscience or Catalog No. WS38665, Waterstone Technology) as indicatedin Table 7 via oral gavage into jugular vein-cannulated male SpragueDawley rats (4 per group) that had been fasted for 16-18 hr prior tooral dosing. At specified time points, blood samples were drawn,harvested for plasma via centrifugation at 5,400 rpm at 4° C. for 5 min,and 100 μl plasma transferred from each sample into a fresh tubecontaining 2 μl of 50% formic acid. The tubes were vortexed for 5-10seconds, immediately placed in dry ice and then stored in a −80° C.freezer until analysis by HPLC/MS.

Table 7 and FIG. 8 provide amphetamine exposure results for ratsadministered with Compound AM-2 and with or without a co-dose of trypsininhibitor. Results in Table 7 are reported, for each group of 4 rats, as(a) maximum plasma concentration (Cmax) of amphetamine (AMP)(average±standard deviation) and (b) time after administration ofCompound AM-2, to reach maximum amphetamine concentration (Tmax)(average±standard deviation).

TABLE 7 Cmax and Tmax values of amphetamine in rat plasma AM-2 AM-2Compound Compound AMP Dose, Dose, 109 Dose, 109 Dose, Cmax ± Tmax ±mg/kg μmol/kg mg/kg μmol/kg sd, ng/mL sd, hr 6 14.5 0 0 50.6 ± 6.3 2.0 ±0.0 6 14.5 30 55 0.91 ± 1.1 12.3 ± 17   Lower limit of quantitation was1.000 ng/mL.

FIG. 8 compares mean plasma concentrations over time of amphetaminerelease following PO administration of Compound AM-2 with or without aco-dose of trypsin inhibitor.

The results in Table 7 and FIG. 8 indicated Compound 109's ability toattenuate Compound AM-2's ability to release amphetamine both bysuppressing Cmax and by delaying Tmax.

Example 12: Pharmacokinetics of Compound AM-2 Following IVAdministration to Rats

This Example compares the plasma concentrations of prodrug andamphetamine in rats following intravenous (IV) administration ofCompound AM-2.

Compound AM-2 (which can be prepared as described in Examples herein)was dissolved in saline and injected into the tail vein of 4 jugularvein-cannulated male Sprague Dawley rats at a dose of 6 mg/kg. Atspecified time points, blood samples were drawn, harvested for plasmavia centrifugation at 5,400 rpm at 4° C. for 5 min, and 100 μl plasmatransferred from each sample into a fresh tube containing 2 μl of 50%formic acid. The tubes were vortexed for 5-10 seconds, immediatelyplaced in dry ice and then stored in a −80° C. freezer until analysis byhigh performance liquid chromatography/mass spectrometry (HPLC/MS).

Table 8 and FIG. 9 provide Compound AM-2 and amphetamine exposureresults for the group of rats administered Compound AM-2 intravenously.Results in Table 8 are reported as maximum plasma concentration (Cmax)values (average±standard deviation) of Compound AM-2 and amphetamine,respectively.

TABLE 8 Cmax values of Compound AM-2 and amphetamine in rat plasma AM-2AM-2 AM-2 AMP Dose, Dose, Cmax ± Cmax ± Compound mg/kg μmol/kg sd,ng/mL* sd, ng/mL{circumflex over ( )} AM-2 6 14.5 2600 ± 260 1.41 ± 0.4*Lower limit of quantitation was 0.100 ng/mL {circumflex over ( )}Lowerlimit of quantitation was 0.500 ng/mL

FIG. 9 compares mean plasma concentrations over time of Compound AM-2and amphetamine following IV administration of Compound AM-2 to rats.

Table 8 and FIG. 9 demonstrate that the plasma concentration ofamphetamine in rats administered Compound AM-2 intravenously was only0.05% of the plasma concentration of Compound AM-2, indicating that IVadministration of Compound AM-2 did not lead to significant release ofamphetamine.

Example 13: In Vivo Tolerability of Compound AM-2 in Rats

This Example demonstrates that Compound AM-2 was tolerated whenadministered intravenously to rats.

Male naïve Sprague-Dawley rats, 4 per dose, were used in the study. Ratswere weighed, and then placed under a heat lamp for 15-20 minutes todilate the lateral tail veins. Dose volumes are based on the bodyweights (1 mL/kg); dosing of Compound AM-2 (which can be prepared asdescribed in Examples herein) was as indicated in Table 9. Beforedosing, rats were placed in Broome restrainers and the drug wasintroduced into one of the tail veins using a syringe and needle. Afterdosing, the timer was set and rats were observed for clinical signs.Blood samples were collected 5 minutes post-dose via the saphenous vein.The rats were observed up to 24 hours. Results are shown in Table 9.

TABLE 9 In vivo tolerability of Compound AM-2 in rats Dose, Dose, Numberof Compound mg/kg μmol/kg Rats dosed Clinical observations AM-2 64 155 4Appeared normal immediately post dose, followed by hyperactivity at 20min. Recovered after 10 min.

The results in Table 9 indicated that rats tolerated a dose of 155μmol/kg of Compound AM-2 and recovered to normal activity within 30minutes.

Example 14: Pharmacokinetics of Compound AM-3 Following POAdministration to Rats

This Example compares the oral (PO) pharmacokinetics (PK) oflisdexamphetamine to PO PK of Compound AM-1 and Compound AM-2, which areembodiments of compounds disclosed and claimed herein.

Saline solutions of Compound AM-3 (lisdexamphetamine) were dosed asindicated in Table 10 using methods as described in Examples 6 and 10above. Samples were collected and stored as described in Examples 6 and10 above.

Table 10 and FIG. 10 provide prodrug and amphetamine exposure resultsfor rats administered Compound AM-3. Results in Table 10 are reported,for each group of 4 rats, as (a) maximum plasma concentration (Cmax) ofprodrug Compound AM-3 (average±standard deviation), (b) time afteradministration of Compound AM-3 to reach maximum prodrug Compound AM-3concentration (Tmax) (average±standard deviation), (c) maximum plasmaconcentration (Cmax) of amphetamine (AMP) (average±standard deviation)and (d) time after administration of Compound AM-3 to reach maximumamphetamine concentration (Tmax) (average±standard deviation).

TABLE 10 Cmax and Tmax values of prodrug Compound AM-3 and amphetaminein rat plasma Prodrug Prodrug AM-3 AM-3 AMP Dose, Dose Cmax ± Tmax ±Cmax ± AMP Com- mg/ μmol/ sd, sd, sd, Tmax ± pound kg kg ng/mL hr ng/mLsd, hr AM-3 22 65 357{circumflex over ( )} ± 133 0.625 ± 0.25 117 ± 45*1.25 ± 0.5 *Lower limit of quantitation was 0.0500 ng/mL {circumflexover ( )}Lower limit of quantitation was 0.500 ng/mL

FIG. 10 compares mean plasma concentrations over time of prodrugdisappearance and of amphetamine release following PO administration ofCompound AM-3.

The results in Table 10 and FIG. 10 indicated that, when both compoundswere administered orally at 65 μmol/kg, the mean concentration ofprodrug Compound AM-3 in plasma was significantly higher than that ofCompound AM-1 (see Example 6).

Example 15: Pharmacokinetics of Compound AM-3 Following IVAdministration to Rats

This Example compares the intravenous (IV) pharmacokinetics (PK) oflisdexamphetamine to IV PK of Compound AM-1 and Compound AM-2, which areembodiments of compounds disclosed and claimed herein.

Saline solutions of Compound AM-3 (lisdexamphetamine) were dosed asindicated in Table 11 using methods as described in Examples 8 and 12above. Samples were collected and stored as described in Examples 8 and12 above.

Table 11 and FIG. 11 provide Compound AM-3 and amphetamine exposureresults for the group of rats administered Compound AM-3 intravenously.Results in Table 11 are reported as maximum plasma concentration (Cmax)values (average±standard deviation) of Compound AM-1 and amphetamine,respectively.

TABLE 11 Cmax values of Compound AM-3 and amphetamine in rat plasma AM-3AMP AM-3 AM-3 Cmax ± Cmax ± Dose, Dose, sd, sd, Compound mg/kg μmol/kgng/mL* ng/mL{circumflex over ( )} AM-3 4.5 13 1670 ± 580 128 ± 4.3*Lower limit of quantitation was 0.0500 ng/mL {circumflex over ( )}Lowerlimit of quantitation was 0.500 ng/mL

FIG. 11 compares mean plasma concentrations over time of Compound AM-3and amphetamine following IV administration of Compound AM-3 to rats.

Table 11 and FIG. 11 demonstrate that the plasma concentration ofamphetamine in rats administered Compound AM-3 intravenously was 7.66%of the plasma concentration of Compound AM-3, indicating that IVadministration of Compound AM-3 led to significantly more amphetaminerelease in the plasma than did IV administration of similar doses ofcompounds of the disclosure (see, for example, Examples 8 and 12).

Example 16: In Vivo Tolerability of Compound AM-3 in Rats

This Example compares the intravenous (IV) tolerability oflisdexamphetamine to the IV tolerability of Compound AM-1 and ofCompound AM-2, which are embodiments of compounds disclosed and claimedherein.

The protocol to assess the IV tolerability of Compound AM-3(lisdexamphetamine) was as described in Examples 9 and 13 above. Resultsare shown in Table 12.

TABLE 12 In vivo tolerability of Compound AM-3 in rats Number Dose,Dose, of Rats Compound mg/kg μmol/kg dosed Clinical observations AM-3 64190 1 Died 10 sec post dose AM-3 32 95 2 Tremors, cyanotic up to 2 min,piloerection at 6 min; 2nd rat: tremors, apnea, died at 40 sec AM-3 1648 4 intermittent tremors at 30 sec, hyperactive at 6 min, repetitivesniffing at 15- 30 min, normal at 2 h

The results in Table 12 indicated that rats tolerated a dose of 48μmol/kg Compound AM-3 and recovered to normal activity by 2 hours.Higher doses of 95 μmol/kg and 190 μmol/kg Compound AM-3 caused death in50% or 100%, respectively, of the rats within less than 1 min. Incomparison, rats were more tolerant to compounds of the disclosure. Forexample, rats tolerated 87 μmol/kg of Compound AM-1 and recovered tonormal activity within 2 minutes (see Example 9), and rats tolerated adose of 155 μmol/kg of Compound AM-2 and recovered to normal activitywithin 30 minutes (see Example 13).

Example 17. Oral Administration of Compound AM-1 Co-Dosed with a TrypsinInhibitor to Rats

This Example demonstrates the ability of a trypsin inhibitor to affectthe ability of an amphetamine prodrug of the embodiments to releaseamphetamine into plasma when such amphetamine prodrug wasco-administered with increasing amounts of trypsin inhibitor orally torats.

Saline solutions of prodrug Compound AM-1 (which can be prepared asdescribed in the example herein) was co-dosed orally to rats withincreasing concentrations of trypsin inhibitor Compound 109 (Catalog No.3081, Tocris Bioscience, or Catalog No. WS38665, Waterstone Technology)as indicated in Table 13, using a method similar to that described inExample 6. Sampling and analysis procedures were also similar to thosedescribed in Example 6.

Table 13 and FIG. 12 provide amphetamine exposure results for ratsadministered 0.6 mg/kg (1.6 μmol/kg) of Compound AM-1 co-dosed withincreasing amounts of trypsin inhibitor Compound 109. Amphetamine Cmaxand Tmax values in Table 13 are reported, for each group of four rats,as described in Example 6.

TABLE 13 Cmax and Tmax values of amphetamine in rat plasma Com- Com-AM-1 AM-1 pound pound AMP Dose, Dose, 109 Dose, 109 Dose, Cmax ± Tmax ±mg/kg μmol/kg mg/kg μmol/kg sd, ng/mL sd, h 0.6 1.6 0 0 5.83 ± 0.42 1.00± 0.0 0.6 1.6 0.1 0.19 5.46 ± 1.6   1.75 ± 0.50 0.6 1.6 0.25 0.46 4.75 ±0.36 2.00 ± 0.0 0.6 1.6 0.5 0.93 3.26 ± 0.56 2.00 ± 0.0 Lower limit ofquantitation was 0.500 ng/mL

FIG. 12 compares mean plasma concentrations over time of amphetaminerelease following PO administration of 0.6 mg/kg (1.6 μmol/kg) ofprodrug Compound AM-1 with increasing amounts of co-dosed trypsininhibitor Compound 109 to rats.

The results in Table 13 and FIG. 12 indicate Compound 109's ability toattenuate release of amphetamine by prodrugs of the embodiments, asindicated by suppressed Cmax and/or delayed Tmax values.

Example 18: Oral Administration of a Single Dose Unit and of MultipleDose Units of a Composition Comprising Prodrug Compound AM-1 and TrypsinInhibitor Compound 109 to Rats

This Example demonstrates the effect of oral administration of singleand multiple dose units comprising prodrug Compound AM-1 and trypsininhibitor Compound 109 to rats.

Saline solutions of Compound AM-1 (which can be prepared as described inthe examples herein) were dosed orally to rats (4 rats per group) atincreasing concentrations ranging from 0.6 to 6 mg/kg (from 1.6 to 16μmol/kg), wherein a single dose was represented as 0.6 mg/kg (1.6μmol/kg) Compound AM-1 in the absence of trypsin inhibitor.

A second set of rats (4 rats per group) were co-dosed with prodrugCompound AM-1 and trypsin inhibitor Compound 109 (Catalog No. 3081,Tocris Bioscience, or Catalog No. WS38665, Waterstone Technology) asdescribed below and indicated in Table 14. Specifically, a salinesolution of a composition comprising 0.6 mg/kg (1.6 μmol/kg) CompoundAM-1 and 0.1 mg/kg (0.2 μmol/kg) Compound 109, representative of asingle dose unit, was administered via oral gavage to each of 4 rats. Itis to be noted that the mole-to-mole ratio of trypsininhibitor-to-prodrug (109-to-AM-1) is 0.12-to-1 as such this dose unitis referred to herein as a 109-to-AM-1 (0.12-to-1) dose unit. Salinesolutions representative of 2 dose units, 3 dose units, 4 dose units, 6dose units, 8 dose units, and 10 dose units (i.e., as indicated in Table14) of the 109-to-AM-1 (0.12-to-1) dose unit were similarly administeredto additional groups of 4 rats.

All rats were jugular vein-cannulated male Sprague Dawley rats that hadbeen fasted for 16-18 h prior to oral dosing. Dosing, sampling andanalysis procedures were similar to those described in Example 6.

Table 14 (top half) and FIG. 13A provide amphetamine exposure results inplasma for rats administered 1, 2, 3, 4, 6, 8 and 10 doses of CompoundAM-1 in the absence of trypsin inhibitor. Table 14 (bottom half) andFIG. 13B provide amphetamine exposure results in plasma for ratsadministered 1, 2, 3, 4, 6, 8 and 10 dose units of the 109-to-AM-1(0.12-to-1) dose unit. Amphetamine Cmax and Tmax values are reported asdescribed in Example 6.

TABLE 14 Cmax and Tmax values of amphetamine in rat plasma AM-1 AM-1 109109 Dose, Dose, Dose, Dose, AMP Amount mg/ μmol/ mg/ μmol/ Cmax ± Tmax ±(Multiple) kg kg kg kg sd, ng/mL sd, h 1 AM-1 dose  0.6 1.6 0   0^(#) 5.83 ± 0.42 1.00 ± 0.00 2 AM-1 doses 1.2 3.2 0   0* 13.2 ± 3.3 1.25 ±0.50 3 AM-1 doses 1.8 4.9 0   0* 17.2 ± 3.9 1.50 ± 0.58 4 AM-1 doses 2.46.5 0   0* 17.6 ± 4.1 1.50 ± 0.58 6 AM-1 doses 3.6 9.7 0   0* 26.0 ± 5.51.75 ± 0.50 8 AM-1 doses 4.8 13 0   0* 25.2 ± 2.3 1.00 ± 0.00 10 AM-1 616 0   0{circumflex over ( )} 64.2 ± 10 1.00 ± 0.00 doses 1 dose unit 0.6 1.6 0.1 0.2^(#) 5.46 ± 1.6 1.75 ± 0.50 2 dose units 1.2 3.2 0.2 0.4*7.45 ± 1.4 2.25 ± 0.50 3 dose units 1.8 4.9 0.3 0.6* 15.4 ± 2.1 2.50 ±0.58 4 dose units 2.4 6.5 0.4 0.7* 13.7 ± 3.3 3.00 ± 0.00 6 dose units3.6 9.7 0.6 1.1* 20.0 ± 6.7 2.75 ± 0.50 8 dose units 4.8 13 0.8 1.5*28.8 ± 6.4 2.75 ± 0.50 10 dose units  6 16 1 1.9^(#) 24.3 ± 9.3 4.00 ±1.2  *Lower limit of quantitation was 0.100 ng/mL {circumflex over( )}Lower limit of quantitation was 1.00 ng/mL ^(#)Lower limit ofquantitation was 0.500 ng/mL

FIG. 13A compares mean plasma concentrations over time of amphetaminerelease following PO administration to rats of a single dose and ofmultiple doses of Compound AM-1 dosed in the absence of trypsininhibitor.

FIG. 13B compares mean plasma concentrations over time of amphetaminerelease following PO administration to rats of a single dose unit and ofmultiple dose units of a composition comprising prodrug Compound AM-1and trypsin inhibitor Compound 109.

The results in Table 14, FIG. 13A and FIG. 13B indicate thatadministration of multiple dose units (as exemplified by 1, 2, 3, 4, 6,8 and 10 dose units of the 109-to-AM-1 (0.12-to-1) dose unit) results ina plasma amphetamine concentration-time PK profile that is not doseproportional to the plasma amphetamine concentration-time PK profile ofthe single dose unit. In addition, the PK profile of the multiple doseunits was modified compared to the PK profile of the equivalent dosageof prodrug in the absence of trypsin inhibitor.

Example 19. Pharmacokinetics of Compound AM-5 Following POAdministration to Rats

This Example indicates the release of amphetamine into plasma when anamphetamine prodrug of the embodiments was administered orally (PO) torats.

Compound AM-5 (which can be prepared as described herein) was dissolvedin water and administered to rats as indicated in Table 15 using methodsas described in Example 6. Samples were collected and stored asdescribed in Example 6.

Table 15 and FIG. 14 provide amphetamine exposure results for ratsadministered Compound AM-5. Amphetamine Cmax, and Tmax values in Table15 are reported for the group of four rats as described in Example 6.

TABLE 15 Cmax and Tmax values amphetamine in rat plasma AMP AMP Dose,Dose Cmax ± Tmax ± Compound mg/kg μmol/kg sd, ng/mL sd, hr AM-5 22 64198{circumflex over ( )} ± 78 2.00 ± 0.0 {circumflex over ( )}Lowerlimit of quantitation was 1.00 ng/mL

FIG. 14 demonstrates the mean plasma concentration over time ofamphetamine release following PO administration to rats of CompoundAM-5.

The results in Table 15 and FIG. 14 indicate that oral administration ofCompound AM-5 leads to release of amphetamine by an amphetamine prodrugof the embodiments.

Example 20. Pharmacokinetics of Compound AM-4 Following POAdministration to Rats

This Example indicates the release of amphetamine into plasma whenCompound AM-4 was administered orally (PO) to rats.

Compound AM-4 (which can be prepared as described herein) was dissolvedin water and administered to rats as indicated in Table 16 using methodsas described in Example 6. Samples were collected and stored asdescribed in Example 6.

Table 16 and FIG. 15 provide amphetamine exposure results for ratsadministered Compound AM-4. Amphetamine Cmax, and Tmax values in Table16 are reported for the group of four rats as described in Example 6.

TABLE 16 Cmax and Tmax values of amphetamine in rat plasma AMP AMP Dose,Dose Cmax ± Tmax ± Compound mg/kg μmol/kg sd, ng/mL sd, hr AM-4 24 631.92 ± 0.20* 1.00 ± 0.0 *Lower limit of quantitation was 0.0500 ng/mL

FIG. 15 demonstrates the mean plasma concentration over time ofamphetamine release following PO administration to rats of CompoundAM-4.

The results in Table 16 and FIG. 15 indicate that oral administration ofCompound AM-4 leads to only minimal release of amphetamine compared tothe release of amphetamine upon oral administration of amphetamineprodrugs of the embodiments.

Example 21. Pharmacokinetics of Compound AM-5 Following IVAdministration to Rats

This Example compares the plasma concentrations of prodrug andamphetamine in rats following intravenous (IV) administration of anamphetamine prodrug of the embodiments.

Compound AM-5 (which can be prepared as described in the examplesherein) was dissolved in water and injected into the tail vein of 4jugular vein-cannulated male Sprague Dawley rats at a dose of 4.5 mg/kg.At specified time points, blood samples were collected, treated, andanalyzed in a manner similar to that described in Example 8.

Table 17 and FIG. 16 provide Compound AM-5 and amphetamine exposureresults for the group of rats administered Compound AM-5 intravenously.Results in Table 17 are reported as maximum plasma concentration (Cmax)values (average±standard deviation) of Compound AM-5 and amphetamine,respectively.

TABLE 17 Cmax values of Compound AM-5 and amphetamine in rat plasma AM-5AM-5 AM-5 AMP Dose, Dose, Cmax ± Cmax ± Compound mg/kg μmol/kg sd,ng/mL{circumflex over ( )} sd, ng/mL* AM-5 4.5 13 2150 ± 1700 6.83 ± 2.0*Lower limit of quantitation was 1.00 ng/mL {circumflex over ( )}Lowerlimit of quantitation was 0.0500 ng/mL

FIG. 16 compares mean plasma concentrations over time of Compound AM-5and amphetamine following IV administration of Compound AM-5 to rats.

Table 17 and FIG. 16 demonstrate that the plasma concentration ofamphetamine in rats administered Compound AM-5 intravenously is only0.32% of the plasma concentration of Compound AM-5, indicating that IVadministration of Compound AM-5, an amphetamine prodrug of theembodiments, does not lead to significant release of amphetamine.

Example 22. Pharmacokinetics of Compounds AM-4 and Compound AM-6Following IV Administration to Rats

This Example demonstrates the plasma concentration of amphetamine inrats following intravenous (IV) administration of Compound AM-4 orCompound AM-6.

Compound AM-4 and Compound AM-6 (each of which can be prepared asdescribed in the examples herein) were each dissolved in saline andinjected into the tail vein of a group of 4 jugular vein-cannulated maleSprague Dawley rats at the respective doses indicated in Table 18. Atspecified time points, blood samples were collected, treated, andanalyzed in a manner similar to that described in Example 8.

Table 18 provides amphetamine exposure results for the group of ratsadministered Compound AM-4 and Compound AM-6 intravenously. Results inTable 18 are reported as maximum plasma concentration (Cmax) values(average±standard deviation) of amphetamine.

TABLE 18 Cmax values of amphetamine in rat plasma AM-6 AM-6 AMP Dose,Dose, Cmax ± Compound mg/kg μmol/kg sd, ng/mL* AM-4 4.5 12 17.9 ± 3.5AM-6 4.8 13 134 ± 36 *Lower limit of quantitation was 1.00 ng/mL

Table 18 demonstrates the release of amphetamine into the plasma of ratsadministered Compound AM-4 or Compound AM-6 intravenously.

Synthesis of Small Molecule Trypsin Inhibitors

Example 23 Synthesis of (S)-ethyl4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazine-1-carboxylate(Compound 101)

Synthesis of4-[(S)-5-([Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl]-amino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-pentanoyl]-piperazine-1-carboxylicacid tert-butyl ester (A)

To a solution of Fmoc-Arg(Pbf)-OH 1 (25.0 g, 38.5 mmol) in DMF (200 mL)at room temperature was added DIEA (13.41 mL, 77.1 mmol). After stirringat room temperature for 10 min, the reaction mixture was cooled to ˜5°C. To the reaction mixture was added HATU (16.11 g, 42.4 mmol) inportions and stirred for 20 min and a solution oftert-butyl-1-piperazine carboxylate (7.18 g, 38.5 mmol) in DMF (50 mL)was added dropwise. The reaction mixture was stirred at ˜5° C. for 5min. The mixture reaction was then allowed to warm to room temperatureand stirred for 2 h. Solvent was removed in vacuo and the residue wasdissolved in EtOAc (500 mL), washed with water (2×750 mL), 1% H₂SO₄ (300mL) and brine (750 mL). The organic layer was separated, dried overNa₂SO₄ and solvent removed in vacuo to a total volume of 100 mL.Compound A was taken to the next step as EtOAc solution (100 mL). LC-MS[M+H] 817.5 (C₄₃H₅₆N₆O₈S+H, calc: 817.4).

Synthesis of4-[(S)-2-Amino-5-({amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-pentanoyl]-piperazine-1-carboxylicacid tert-butyl ester (B)

To a solution of compound A (46.2 mmol) in EtOAc (175 mL) at roomtemperature was added piperidine (4.57 mL, 46.2 mmol) and the reactionmixture was stirred for 18 h at room temperature. Next the solvent wasremoved in vacuo and the resulting residue dissolved in minimum amountof EtOAc (˜50 mL) and hexane (˜1 L) was added. The precipitated crudeproduct was filtered off and recrystallised again with EtOAc (˜30 mL)and hexane (˜750 mL). The precipitate was filtered off, washed withhexane and dried in vacuo to afford compound B (28.0 g, 46.2 mmol).LC-MS [M+H] 595.4 (C₂₈H₄₆N₆O₆S+H, calc: 595.3). Compound B was usedwithout further purification.

Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperazine-1-carboxylicacid tert-butyl ester (C)

To a solution of compound B (28.0 g, 46.2 mmol) in THF (250 mL) wasadded aqueous 1N NaOH (171 mL). The reaction mixture was cooled to ˜5°C., a solution of 2-naphthalene sulfonylchloride (26.19 g, 115.6 mmol)in THF (125 mL) was added dropwise. The reaction mixture was stirred at˜5° C. for 10 min, with stirring continued at room temperature for 2 h.The reaction mixture was diluted with EtOAc (1 L), washed with aqueous1N NaOH (1 L), water (1 L) and brine (1 L). The organic layer wasseparated, dried over Na₂SO₄ and removal of the solvent in vacuo toafford compound C (36.6 g, 46.2 mmol). LC-MS [M+H] 785.5(C₃₈H₅₂N₆O₈S2+H, calc: 785.9). Compound C was used without furtherpurification.

Synthesis of 2,2,4,6,7-Pentamethyl-2,3-dihydro-benzofuran-5-sulfonicacid1-amino-1-[(S)-4-(naphthalene-2-sulfonylamino)-5-oxo-5-piperazin-1-yl-pentylamino]-meth-(E)-ylideneamide(D)

To a solution of compound C (36.6 g, 46.2 mmol) in dioxane (60 mL) wasadded 4M HCl in dioxane (58 mL) dropwise. The reaction mixture wasstirred at room temperature for 1.5 h. Et₂O (600 mL) was added to thereaction mixture, the precipitated product was filtered off, washed withEt₂O and finally dried in vacuo to afford compound D (34.5 g, 46.2mmol). LC-MS [M+H] 685.4 (C₃₃H₄₄N₆O₆S2+H, calc: 685.9). Compound D wasused without further purification.

Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperazine-1-carboxylicacid ethyl ester (E)

To a solution of compound D (8.0 g, 11.1 mmol) in CHCl₃ (50 mL) wasadded DIEA (4.1 mL, 23.3 mmol) at room temperature and stirred for 15min. The mixture was cooled to ˜5° C., ethyl chloroformate (1.06 mL,11.1 mmol) was added dropwise. After stirring at room temperatureovernight (˜18 h), solvent removed in vacuo. The residue was dissolvedin MeOH (˜25 mL) and Et₂O (˜500 mL) was added. The precipitated crudeproduct was filtered off, washed with Et₂O and dried in vacuo to affordcompound E (8.5 g, 11.1 mmol). LC-MS [M+H]757.6 (C₃₆H₄₈N₆O₈S2+H, calc:757.9). Compound E was used without further purification.

Synthesis of (S)-ethyl4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazine-1-carboxylate(Compound 101)

A solution of 5% m-cresol/TFA (50 mL) was added to compound E (8.5 g,11.1 mmol) at room temperature. After stirring for 1 h, the reactionmixture was precipitated with Et₂O (˜500 mL). The precipitate wasfiltered and washed with Et₂O and dried in vacuo to afford the crudeproduct. The crude product was purified by preparative reverse phaseHPLC. [Column: VARIAN, LOAD & LOCK, L&L 4002-2, Packing: Microsorb100-10 C18, Injection, Volume: ˜15 mL×2, Injection flow rate: 20 mL/min,100% A, (water/0.1% TFA), Flow rate: 100 mL/min, Fraction: 30 Sec (50mL), Method: 0% B (MeCN/0.1% TFA)-60% B/60 min/100 mL/min/254 nm].Solvents were removed from pure fractions in vacuo. Trace of water wasremoved by co-evaporation with 2×i-PrOH (50 mL). The residue wasdissolved in a minimum amount of i-PrOH and product was precipitatedwith 2 M HCl in Et₂O. Product was filtered off and washed with Et₂O anddried in vacuo to afford Compound 101 as HCl salt 7 (3.78 g, 63% yield,99.4% purity). LC-MS [M+H] 505.4 (C₃₈H₅₂N₆O₈S2+H, calc: 505.6).

Example 24 Synthesis of (S)-ethyl4-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperazine-1-carboxylate(Compound 101)

Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-tert-butoxycarbonylamino-pentanoyl]-piperazine-1-carboxylicacid ethyl ester (F)

To a solution of Boc-Arg(Pbf)-OH (13.3 g, 25.3 mmol) in DMF (10 mL) wasadded DIEA (22.0 mL, 126.5 mmol) at room temperature and stirred for 15min. The reaction mixture was then cooled to ˜5° C. and HATU (11.5 g,30.3 mmol) was added in portions and stirred for 30 min, followed by thedropwise addition of ethyl-1-piperazine carboxylate (4.0 g, 25.3 mmol)in DMF (30 mL). After 40 min, the reaction mixture was diluted withEtOAc (400 mL) and poured into H₂O (1 L). Extracted with EtOAc (2×400mL) and washed with H₂O (800 mL), 2% H₂SO₄ (500 mL), H₂O (2×800 mL) andbrine (800 mL). Organic layer was separated, dried over MgSO₄ andsolvent removed in vacuo. The resultant oily residue was dried in vacuoto afford compound F (16.4 g, 24.5 mmol) as foamy solid. LC-MS [M+H]667.2 (C₃₁H₅₀N₆O₈S+H, calc: 667.8). Compound F was used without furtherpurification.

Synthesis of4-[(S)-2-Amino-5-({amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5sulfonylimino]-methyl}-amino)-pentanoyl]-piperazine-1-carboxylic acidethyl ester (G)

A solution of compound F (20.2 g, 30.2 mmol) in dichloromethane (90 mL)was treated with 4.0 N HCl in 1,4-dioxane (90 mL, 363.3 mmol) andstirred at room temperature for 2 h. Next most of the dichloromethane(˜90%) was removed in vacuo and Et₂O (˜1 L) was added. The resultantprecipitate was filtered off and washed with Et₂O and dried in vacuo toafford compound G (17.8 g, 30.2 mmol). LC-MS [M+H] 567.8 (C₂₆H₄₂N₆O₆S+H,calc: 567.8). Compound G was used without further purification.

Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-(2,4,6-triisopropyl-benzenesulfonylamino)-pentanoyl]-piperazine-1-carboxylicacid ethyl ester (H)

To a solution of compound G (1.0 g, 1.8 mmol) in THF (7 mL) was added3.1N aqueous NaOH (4.0 mL) and stirred for 5 min. The reaction mixturewas cooled to ˜5° C., and then a solution of tripsyl chloride addeddropwise (2.2 g, 7.3 mmol) in THF (5 mL) and stirred at room temperatureovernight (˜18 h). The reaction mixture was diluted with H₂O (130 mL),acidified with 2% H₂SO₄ (15 mL) and extracted with EtOAc (3×80 mL).Organic layer were combined and washed with H₂O (2×400 mL), saturatedNaHCO₃ (100 mL), H₂O (200 mL) and brine (200 mL). The organic layer wasseparated, dried over MgSO₄ and solvent removed in vacuo to afford (2.9g) of crude product. This was purified by normal phase flashchromatography (5-10% MeOH/DCM) to afford compound H (0.52 g, 1.0 mmol).LC-MS [M+H] 833.8 (C₄₁H₆₄N₆O₈S₂+H, calc: 834.1).

Synthesis of (S)-ethyl4-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperazine-1-carboxylate(Compound 102)

A solution of 5% m-cresol/TFA (40 mL) was added to compound H (3.73 g,3.32 mmol) at room temperature. After stirring for 45 min, solvents wereremoved in vacuo. Residue was dissolved in dichloromethane (100 mL),washed with H₂O (3×200 mL) and brine (200 mL). The organic layer wasseparated, dried over MgSO₄ and then the solvent removed in vacuo. Theresidue was dissolved in dichloromethane (˜5 mL) and then hexane (˜250mL) was added and a precipitate was formed. This was washed with hexaneand dried in vacuo to afford the crude product (1.95 g). The crudeproduct was purified by reverse phase HPLC [Column: VARIAN, LOAD & LOCK,L&L 4002-2, Packing: Microsorb 100-10 C18, Injection Volume: ˜15 mL,Injection flow rate: 20 mL/min, 100% A, (water/0.1% TFA), Flow rate: 100mL/min, Fraction: 30 Sec (50 mL), Method: 25% B (MeCN/0.1% TFA)/70% B/98min/100 mL/min/254 nm]. Solvents were removed from pure fractions invacuo. Trace of water was removed by co-evaporation with 2× i-PrOH (50mL). The residue was dissolved in a minimum amount of i-PrOH and productwas precipitated with 2 M HCl in Et₂O. Product was filtered off andwashed with Et₂O and dried in vacuo to afford the product as HCl salt ofCompound 102 (0.72 g, 35% yield, 99.8% purity). LC-MS [M+H] 581.6(C₂₈H₄₈N₆O₅S+H, calc: 581.7).

Example 25 Synthesis of (S)-ethyl1-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperidine-4-carboxylateHCl salt (Compound 103)

Synthesis of 1-[boc-Arg(Pbf)]-piperidine-4-carboxylic acid ethyl ester(I)

To a solution of Boc-Arg(Pbf)-OH (3.4 g, 6.36 mmol) and HATU (2.9 g,7.63 mmol) in DMF (15 mL) was added DIEA (7.4 mL, 42.4 mmol) and thereaction mixture was stirred for 10 min at room temperature. A solutionof ethyl isonipecotate (1.0 g, 6.36 mmol) in DMF (6 mL) was added to thereaction mixture dropwise. The reaction mixture was stirred at roomtemperature for 1 h, then diluted with EtOAc (150 mL) and poured intowater (500 mL). The product was extracted with EtOAc (2×100 mL). Theorganic layer was washed with aqueous 0.1 N HCl (200 mL), 2% aqueoussodium bicarbonate (200 mL), water (200 mL) and brine (200 mL). Theorganic layer was then dried over sodium sulfate, filtered, and thenevaporated in vacuo. The resultant oily product was dried in vacuoovernight to give compound I (3.7 g, 5.57 mmol) as a viscous solid.LC-MS [M+H] 666.5 (C₃₂H₅₁N₅O₈S+H, calc: 666.7). Compound I was usedwithout further purification.

Synthesis of 1-[Arg(Pbf)]-piperidine-4-carboxylic acid ethyl ester HClsalt (J)

To a solution of compound I (4.7 g, 7.07 mmol) in dichloromethane (25mL) was added 4N HCl in dioxane (25.0 mL, 84.84 mmol), and the reactionmixture was stirred at room temperature for 2 h. The reaction mixturewas concentrated in vacuo to ˜20 mL of solvent, and then diluted withdiethyl ether (250 mL) to produce a white fine precipitate. The reactionmixture was stirred for 1 h and the solid was washed with ether (50 mL)and dried in vacuo overnight to give compound J (4.3 g, 7.07 mmol) as afine powder. LC-MS [M+H] 566.5 (C₂₇H₄₃N₅O₆S+H, calc: 566.7). Compound Jwas used without further purification.

Synthesis of1-[5(S)—(N′-Pbf-guanidino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperidine-4-carboxylicacid ethyl ester (K)

To a solution of compound J (1.1 g, 1.6 mmol) and NaOH (260 mg, 5.9mmol) in a mixture of THF (5 mL) and water (3 mL) was added a solutionof 2-naphthalosulfonyl chloride (0.91 g, 2.5 mmol) in THF (10 mL)dropwise with stirring at ˜5° C. The reaction mixture was stirred atroom temperature for 1 h, then diluted with water (5 mL). Aqueous 1N HCl(5 mL) was added to obtain pH ˜3. Additional water was added (20 mL),and the product was extracted with ethyl acetate (3×50 mL). The organiclayer was removed and then washed with 2% aqueous sodium bicarbonate (50mL), water (50 mL) and brine (50 mL). The extract was dried overanhydrous sodium sulfate, filtered, and was evaporated in vacuo. Theformed oily product was dried in vacuo overnight to give compound K (1.3g, 1.6 mmol) as an oily foaming solid. LC-MS [M+H] 756.5(C₃₇H₄₉N₅O₈S₂+H, calc: 756.7). Compound K was used without furtherpurification.

Synthesis of (S)-ethyl1-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperidine-4-carboxylateHCl salt (Compound 103)

To a flask, was added compound K (1.3 g, 1.6 mmol) and then treated with5% m-cresol/TFA (10 mL). The reaction mixture was stirred at roomtemperature for 1 h. Next, the reaction mixture was concentrated invacuo to a volume ˜5 mL. Diethyl ether (200 mL) was then added to theresidue, and formed fine white precipitate. The precipitate was filteredoff and washed with ether (2×25 mL). The resultant solid was dried invacuo overnight to give a crude material, which was purified bypreparative reverse phase HPLC. [Nanosyn-Pack Microsorb (100-10) C-18column (50×300 mm); flow rate: 100 mL/min; injection volume 12 mL(DMSO-water, 1:1, v/v); mobile phase A: 100% water, 0.1% TFA; mobilephase B: 100% ACN, 0.1% TFA; gradient elution from 25% B to 55% B in 90min, detection at 254 nm]. Fractions containing desired compound werecombined and concentrated in vacuo. The residue was dissolved in i-PrOH(50 mL) and evaporated in vacuo (repeated twice). The residue was nextdissolved in i-PrOH (5 mL) and treated with 2 N HCl/ether (100 mL, 200mmol) to give a white precipitate. It was dried in vacuo overnight togive Compound 103 (306 mg, 31% yield, 95.7% purity) as a white solid.LC-MS [M+H] 504.5 (C₂₄H₃₃N₅O₅S+H, calc: 504.6).

Example 26 Synthesis of (S)-ethyl1-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperidine-4-carboxylateHCl salt (Compound 104)

Synthesis of1-[5(S)—(N′-Pbf-guanidino)-2-(2,4,6-triisopropyl-benzenesulfonylamino)-pentanoyl]-piperidine-4-carboxylicacid ethyl ester (N)

To a solution of compound J (1.0 g, 1.6 mmol) and NaOH (420.0 mg, 10.4mmol) in a mixture of THF (5 mL) and water (4 mL) was added a solutionof 2,4,6-triisopropyl-benzenesulfonyl chloride (2.4 g, 8.0 mmol)dropwise with stirring and maintained at ˜5° C. The reaction mixture wasthen stirred at room temperature for 1 h, monitoring the reactionprogress, then diluted with water (20 mL), and acidified with aqueous 1N HCl (5 mL) to pH ˜3. Additional water was added (30 mL), and theproduct was extracted with EtOAc (3×50 mL). The organic layer was washedwith 2% aqueous sodium bicarbonate (50 mL), water (50 mL) and brine (50mL). The organic layer was dried over anhydrous sodium sulfate,filtered, and was evaporated in vacuo. Formed oily residue was dried ina vacuo overnight to give compound N (1.0 g, 1.2 mmol) as an oilymaterial. LC-MS [M+H] 832.8 (C₄₂H₆₅N₅O₈S2+H, calc: 832.7). Compound Nwas used without further purification.

Synthesis of (S)-ethyl1-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperidine-4-carboxylateHCl salt (Compound 104)

To a flask was added compound N (2.3 g, 2.8 mmol) and then treated with5% m-cresol/TFA (16 mL). The reaction mixture was stirred at roomtemperature for 1 h. The reaction mixture was then concentrated in vacuoto a volume of 5 mL. Hexane (200 mL) was added to the residue anddecanted off to give an oily precipitate. The product was purified bypreparative reverse phase HPLC. [Nanosyn-Pack Microsorb (100-10) C-18column (50×300 mm); flow rate: 100 mL/min; injection volume 15 mL(DMSO-water, 1:1, v/v); mobile phase A: 100% water, 0.1% TFA; mobilephase B: 100% ACN, 0.1% TFA; gradient elution from 35% B to 70% B in 90min, detection at 254 nm]. Fractions containing desired compound werecombined and concentrated in vacuo. The residue was dissolved in i-PrOH(100 mL) and evaporated in vacuo (repeated twice). The residue wasdissolved in i-PrOH (5 mL) and treated with 2 N HCl/ether (100 mL, 200mmol) to give an oily residue. It was dried in vacuo overnight to giveCompound 104 (1.08 g, 62.8%) as a viscous solid. LC-MS [M+H] 580.6(C₂₉H₄₉N₅O₅S+H, calc: 580.8).

Example 27 Synthesis of(S)-6-(4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazin-1-yl)-6-oxohexanoicacid (Compound 105)

Synthesis of6-[4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl]-amino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperazin-1-yl}-6-oxo-hexanoicacid methyl ester (O)

To a solution of compound D (1.5 g, 2.08 mmol) in CHCl₃ (50 mL) wasadded DIEA (1.21 mL, 4.16 mmol) followed by adipoyl chloride (0.83 mL,6.93 mmol) dropwise. The reaction mixture was stirred at roomtemperature overnight (˜18 h). Solvents were removed in vacuo and theresidue was dried in vacuo to afford the compound O (2.1 g, amountexceeded quantative). LC-MS [M+H] 827.5 (C₄₀H₅₄N₆O₉S₂+H, calc: 827.3).Compound O was used without further purification.

Synthesis of6-[4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl]-amino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperazin-1-yl}-6-oxohexanoicacid (P)

To a solution of compound 0 (2.1 g, 2.08 mmol) in THF (5 mL), H₂O (5 mL)was added 2 M aq LiOH (6 mL). The reaction mixture was stirred at roomtemperature for 2 h. Solvents were removed in vacuo, then the residuewas dissolved in water (˜50 mL), acidified with saturated aqueous NaHSO₄(˜100 mL) and extracted with EtOAc (2×100 mL). The organic layer wasdried over Na₂SO₄ and removal of the solvent gave compound P (1.72 g,2.08 mmol). LC-MS [M+H] 813.5 (C₃₉H₅₂N₆O₉S₂+H, calc: 813.3). Compound Pwas used without further purification.

Synthesis of(S)-6-(4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazin-1-yl)-6-oxohexanoicacid (Compound 105)

A solution of 5% m-cresol/TFA (25 mL) was added to compound P (1.72 g,2.08 mmol) at room temperature. After stirring for 30 min, the reactionmixture was precipitated with addition of Et₂O (˜200 mL). Theprecipitate was filtered and washed with Et₂O and dried in vacuo toafford the crude product. The crude product was purified by preparativereverse phase HPLC [Column: VARIAN, LOAD & LOCK, L&L 4002-2, Packing:Microsorb 100-10 C18, Injection Volume: ˜25 mL, Injection flow rate: 20mL/min, 95% A, (water/0.1% TFA), Flow rate: 100 mL/min, Fraction: 30 Sec(50 mL), Method: 5% B (MeCN/0.1% TFA)/5 min/25% B/20 min/25% B/15min/50% B/25 min/100 mL/min/254 nm]. Solvents were removed from purefractions in vacuo. Trace amounts of water was removed by co-evaporationwith i-PrOH (25 mL) (repeated twice). The residue was dissolved in aminimum amount of i-PrOH, then 2 M HCl in Et₂O (˜50 mL) was added anddiluted with Et₂O (˜250 mL). Precipitate formed was filtered off andwashed with Et₂O and dried in vacuo to afford the product as HCl saltCompound 105 (0.74 g, 59% yield, 98.9% purity). LC-MS [M+H] 561.4(C₂₆H₃₆N₆O₆S+H, calc: 561.2).

Example 28 Synthesis of 3-(4-carbamimidoylphenyl)-2-oxopropanoic acid(Compound 107)

Compound 107, i.e., 3-(4-carbamimidoylphenyl)-2-oxopropanoic acid can beproduced using methods known to those skilled in the art, such as thatdescribed by Richter P et al, Pharmazie, 1977, 32, 216-220 andreferences contained within. The purity of Compound 107 used herein wasestimated to be 76%, an estimate due low UV absorbance of this compoundvia HPLC. Mass spec data: LC-MS [M+H] 207.0 (C10H10N2O3+H, calc: 207.1).

Example 29 Synthesis of(S)-5-(4-carbamimidoylbenzylamino)-5-oxo-4-((R)-4-phenyl-2-(phenylmethylsulfonamido)butanamido)pentanoicacid (Compound 108)

Preparation 30 Synthesis of(S)-4-tert-butoxycarbonylamino-4-(4-cyano-benzylcarbamoyl)-butyric acidbenzyl ester (Q)

A solution of Boc-Glu(OBzl)-OH (7.08 g, 21.0 mmol), BOP (9.72 g, 22.0mmol) and DIEA (12.18 mL, 70.0 mmol) in DMF (50 mL) was maintained atroom temperature for 20 min, followed by the addition of4-(aminomethyl)benzonitrile hydrochloride (3.38 g, 20.0 mmol). Thereaction mixture was stirred at room temperature for an additional 1 hand diluted with EtOAc (500 mL). The obtained solution was extractedwith water (100 mL), 5% aq. NaHCO₃ (100 mL) and water (2×100 mL). Theorganic layer was dried over MgSO₄, evaporated and dried in vacuo toprovide compound Q (9.65 g, yield exceeded quantitative) as yellowishoil. LC-MS [M+H] 452.0 (C₂₅H₂₉N₃O₅+H, calc: 452.4). Compound Q was usedwithout further purification.

Synthesis of(S)-4-tert-butoxycarbonylamino-4-[4-(N-hydroxycarbamimidoyl)-benzylcarbamoyl]-butyric acid benzyl ester (R)

A solution of compound Q (9.65 g, 20.0 mmol), hydroxylaminehydrochloride (2.10 g, 30.0 mmol) and DIEA (5.22 mL, 30.0 mmol) inethanol (abs., 150 mL) was refluxed for 6 h. The reaction mixture wasallowed to cool to room temperature and stirred for additional 16 h. Thesolvents were evaporated in vacuo. The resultant residue was dried invacuo to provide compound R (14.8 g, yield exceeded quantitative) asyellowish oil. LC-MS [M+H] 485.5 (C₂₅H₃₂N₄O₆+H, calc: 485.8). Compound Rwas used without further purification.

Synthesis of(S)-4-tert-butoxycarbonylamino-4-[4-(N-acetylhydroxycarbamimidoyl)-benzylcarbamoyl]-butyric acid benzyl ester (S)

A solution of compound R (14.8 g, 20.0 mmol) and acetic anhydride (5.7mL, 60.0 mmol) in acetic acid (100 mL) was stirred at room temperaturefor 45 min, and then solvent was evaporated in vacuo. The resultantresidue was dissolved in EtOAc (300 mL) and extracted with water (2×75mL) and brine (75 mL). The organic layer was then dried over MgSO₄,evaporated and dried in vacuo to provide compound S (9.58 g, 18.2 mmol)as yellowish solid. LC-MS [M+H] 527.6 (C₂₇H₃₄N₄O₇+H, calc: 527.9).Compound S was used without further purification.

Synthesis of (S)-4-[4-(N-acetylhydroxycarbamimidoyl)-benzylcarbamoyl]-butyric acid benzyl ester (T)

Compound S (9.58 g, 18.2 mmol) was dissolved in 1,4-dioxane (50 mL) andtreated with 4 N HCl/dioxane (50 mL, 200 mmol) at room temperature for 1h. Next, the solvent was evaporated in vacuo. The resultant residue wastriturated with ether (200 mL). The obtained precipitate was filtrated,washed with ether (100 mL) and hexane (50 mL) and dried in vacuo toprovide compound T (9.64 g, yield exceeded quantitative) as off-whitesolid. LC-MS [M+H]426.9 (C₂₂H₂₆N₄O₅+H, calc: 427.3). Compound T was usedwithout further purification.

Synthesis of (R)-4-phenyl-2-phenylmethanesulfonylamino-butyric acid (U)

A solution of D-homo-phenylalanine (10.0 g, 55.9 mmol) and NaOH (3.35 g,83.8 mmol) in a mixture of 1,4-dioxane (80 mL) and water (50 mL) wascooled to ˜5° C., followed by alternate addition of α-toluenesulfonylchloride (16.0 g, 83.8 mmol; 5 portions by 3.2 g) and 1.12 M NaOH (50mL, 55.9 mmol; 5 portions by 10 mL) maintaining pH>10. The reactionmixture was then acidified with 2% aq. H₂SO₄ to a pH of about pH 2. Theobtained solution was extracted with EtOAc (2×200 mL). The obtainedorganic layer was washed with water (3×75 mL), dried over MgSO₄ and thenthe solvent was evaporated in vacuo. The resultant residue was dried invacuo to provide compound U (12.6 g, 37.5 mmol) as white solid. LC-MS[M+H] 334.2 (C₁₇H₁₉NO₄S+H, calc: 333.4). Compound U was used withoutfurther purification.

Synthesis of(S)-4-[4-(N-acetylhydroxycarbamimidoyl)-benzylcarbamoyl]-4-((R)-4-phenyl-2-phenylmethanesulfonylamino-butyrylamino)-butyricacid benzyl ester (V)

A solution of compound U (5.9 g, 17.8 mmol), compound T di-hydrochloride(18.0 mmol), BOP (8.65 g, 19.6 mmol) and DIEA (10.96 mL, 19.6 mmol) inDMF (250 mL) was stirred at room temperature for 2 h. The reactionmixture was then diluted with EtOAc (750 mL) and extracted with water(200 mL). The formed precipitate was filtrated, washed with EtOAc (200mL) and water (200 mL) and dried at room temperature overnight (˜18 h)to provide compound V (8.2 g, 11.0 mmol) as off-white solid. LC-MS [M+H]743.6 (C₃₉H₄₃N₅O₈S+H, calc: 743.9). Compound V was used without furtherpurification.

Synthesis of(S)-5-(4-carbamimidoylbenzylamino)-5-oxo-4-((R)-4-phenyl-2-(phenylmethylsulfonamido)butanamido)pentanoicacid (Compound 108)

Compound V (8.0 g, 10.77 mmol) was dissolved in acetic acid (700 mL)followed by the addition of Pd/C (5% wt, 3.0 g) as a suspension in water(50 mL). Reaction mixture was subjected to hydrogenation (Parrapparatus, 50 psi H₂) at room temperature for 3 h. The catalyst wasfiltered over a pad of Celite on sintered glass filter and washed withmethanol. Filtrate was evaporated in vacuo to provide Compound 108 ascolorless oil. LC-MS [M+H] 594.2 (C₃₀H₃₅N506S+H, calc: 594). Obtainedoil was dissolved in water (150 mL) and subjected to HPLC purification.[Nanosyn-Pack YMC-ODS-A (100-10) C-18 column (75×300 mm); flow rate: 250mL/min; injection volume 150 mL; mobile phase A: 100% water, 0.1% TFA;mobile phase B: 100% acetonitrile, 0.1% TFA; isocratic elution at 10% Bin 4 min., gradient elution to 24% B in 18 min, isocratic elution at 24%B in 20 min, gradient elution from 24% B to 58% B in 68 min; detectionat 254 nm]. Fractions containing desired compound were combined andconcentrated in vacuo. Residue was dissolved in i-PrOH (75 mL) andevaporated in vacuo (procedure was repeated twice) to provide Compound108 (4.5 g, 70% yield, 98.0% purity) as white solid. LC-MS [M+H] 594.2(C₃₀H₃₅N₅O₆S+H, calc: 594). Retention time*: 3.55 min. *—[ChromolithSpeedRod RP-18e C18 column (4.6×50 mm); flow rate 1.5 mL/min; mobilephase A: 0.1% TFA/water; mobile phase B 0.1% TFA/acetonitrile; gradientelution from 5% B to 100% B over 9.6 min, detection 254 nm]

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A compound of formula AM-1; AM-2; or AM-5:

or a salt, hydrate or solvate thereof.
 2. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a compound of claim
 1. 3. A composition comprising aGI enzyme inhibitor and a compound of formula AM-1; AM-2; or AM-5:

or a salt, hydrate or solvate thereof.
 4. The composition of claim 2,wherein the composition is formulated for oral administration.
 5. Thecomposition of claim 3, wherein the composition is formulated for oraladministration.
 6. The composition of claim 3, wherein the compositioncomprises a compound of formula AM-1 in a dosage amount of from 4 mg/kgto 32 mg/kg.
 7. The composition of claim 3, wherein the compositioncomprises a compound of formula AM-1 in a dosage amount of about 5mg/kg.
 8. The composition of claim 3, wherein the composition comprisesa compound of formula AM-1 in a dosage amount of about 6 mg/kg.
 9. Thecomposition of claim 3, wherein the composition comprises a compound offormulate AM-1 in a dosage amount of about 24 mg/kg.
 10. The compositionof claim 3, wherein the composition comprises a compound of formulateAM-1 in a dosage amount of about 32 mg/kg.
 11. The composition of claim3, wherein the composition comprises a compound of formula AM-2 in adosage amount of from 4 mg/kg to 64 mg/kg.
 12. The composition of claim3, wherein the composition comprises a compound of formula AM-2 in adosage amount of about 6 mg/kg.
 13. The composition of claim 3, whereinthe composition comprises a compound of formulate AM-2 in a dosageamount of about 27 mg/kg.
 14. The composition of claim 3, wherein thecomposition comprises a compound of formulate AM-2 in a dosage amount ofabout 64 mg/kg.
 15. The composition of claim 3, wherein the compositioncomprises a compound of formula AM-5 in a dosage amount of from 4 mg/kgto 27 mg/kg.
 16. The composition of claim 3, wherein the compositioncomprises a compound of formulate AM-5 in a dosage amount of about 4.5mg/kg.
 17. The composition of claim 3, wherein the composition comprisesa compound of formulate AM-5 in a dosage amount of about 22 mg/kg.